Methods for the treatment of gout

ABSTRACT

Disclosed are methods for the treatment and/or prevention of gout, comprising administering to a subject an effective amount of anti-IL-1β antibody or fragment thereof.

This application is a continuation of U.S. application Ser. No.15/419,991, filed Jan. 30, 2017, which is a continuation of U.S.application Ser. No. 14/137,892, filed Dec. 20, 2013, which is acontinuation of U.S. application Ser. No. 12/338,957, filed Dec. 18,2008, now U.S. Pat. No. 8,637,029, which claims benefit under 35 U.S.C.§ 119 of U.S. Provisional Application No. 61/015,633, filed Dec. 20,2007, U.S. Provisional Application No. 61,059,378, filed Jun. 6, 2008,and U.S. Provisional Application No. 61/095,191, filed Sep. 8, 2008, thedisclosure of each of which is incorporated by reference herein in itsentirety.

FIELD OF INVENTION

The invention relates to methods and materials for the treatment and/orprevention of gout. Such methods and materials may be used to treat asubject suffering from gout or to prevent occurrence of the same in anat risk subject.

BACKGROUND OF THE INVENTION

The present disclosure is directed to methods and materials for thetreatment and/or prevention of gout in a subject. Such methods andmaterials may be used to treat a mammalian (e.g., human) subjectsuffering from gout or to prevent occurrence of the same in an at risksubject.

Gout is a form of acute arthritis that causes severe pain and swellingin the joints. Gouty arthritis accounted for an estimated 3.9 millionoutpatient visits in the United States in 2002. Unlike other rheumaticdiseases, the etiology of gout is well characterized, itspathophysiology is well understood, and the disease is easily diagnosed.For many patients, therapy with nonsteroidal anti-inflammatory drugs(NSAIDs) or corticosteroids for acute attacks and prevention ofrecurrence with agents that lower the serum uric acid levels are highlyeffective. However, these therapies are not sufficient for many patientswith acute, chronic or refractory gout due to their lack of adequateclinical efficacy, associated toxicities, or because of co-morbiddiseases.

Gout is precipitation of crystals into tissue, usually in and aroundjoints, most often causing recurrent acute or chronic arthritis.

The disease is marked by deposits of monosodium urate (MSU) crystalsinto tissue, usually in and around the joints and in the synovial fluidand lining, and usually an excessive amount of uric acid in the blood.Intense joint inflammation occurs as white blood cells engulf the uricacid crystals, causing pain, heat, and redness of the joint tissues.Gouty arthritis is due to monosodium urate crystal-induced release ofproinflammatory cytokines from leukocytes. Among the many cytokinesimplicated, IL-1 may have a special role in the inflammatory network, asMSU crystals stimulate IL-1 release by monocytes and synovialmononuclear cells. Acute gout flares usually come on suddenly, go awayafter 5 to 10 days, and can keep recurring.

IL-1β is a pro-inflammatory cytokine secreted by a number of differentcell types including monocytes and macrophages. When released as part ofan inflammatory reaction, IL-1β produces a range of biological effects,mainly mediated through induction of other inflammatory mediators suchas corticotrophin, platelet factor-4, prostaglandin E2 (PGE2), IL-6, andIL-8. IL-1β induces both local and systemic inflammatory effects throughthe activation of the IL-1 receptor found on almost all cell types. Theinterleukin-1 (IL-1) family of cytokines has been implicated in a numberof disease states. IL-1 family members include IL-1α, IL-1β, and IL-1Ra.Although related by their ability to bind to IL-1 receptors (IL-1R1 andIL-1R2), each of these cytokines is different, being expressed by adifferent gene and having a different primary amino acid sequence.Furthermore, the physiological activities of these cytokines can bedistinguished from each other.

Experiments indicating the apparent involvement of IL-1β and otherinflammatory mediators in gout have been published (see for example,Petrilli et al., Joint Bone Spine (2007) 74:571-576; Pope et al.,Arthritis Rheum. (2007) 56:3183-3188; Chen et al., J. Clin. Invest.(2006) 116:2073-2075; Akahoshi, T., et al., Curr. Opin. Rheumatol.(2007) 19:146-150; Martinon, F., et al., Nature (2006) 440:237-241; andCronstein et al., Arthritis Res. Ther. (2006) 8, Suppl. 1:S3). So etal., Arthritis Res. Ther. (2007) 9(2):R28 describe the use of arecombinant IL-1 receptor antagonist (IL-1Ra, anakinra) in an open labelstudy for the treatment of acute gout, performed with daily dosing of100 mg subcutaneously for 3 days. McGonagle, et al., Ann. Rheum. Dis.(2007) 66:1683-1684 describe the use of a recombinant IL-1 receptorantagonist (IL-1Ra, anakinra) for the treatment of gout in a patientreceiving continuous daily subcutaneous doses of 100 mg. The dailydosing of injectable medications is generally undesirable and may resultin problems with patient compliance, thereby decreasing effectiveness ofthis treatment modality/or limiting its desirability. Thus, thereremains a need for effective means to treat gout, particularly treatmentcompositions and methods that do not require frequent (e.g., daily)injections.

Because of the problems with current treatments, new therapies to treatgout are needed to replace or complement available pharmaceuticalapproaches. The present disclosure provides compositions and methods forthe treatment of gout (e.g., acute gout, chronic gout, refractory gout).The methods disclosed herein comprise, for example, administering ananti-IL-1β antibody or fragment thereof. Methods that directly targetthe IL-1β ligand with an antibody, particularly antibodies that exhibithigh affinity, provide advantages over other potential methods oftreatment, such as IL-1β receptor antagonists (e.g., IL-1Ra, Anakinra).The challenge for IL-1 receptor antagonist-based therapeutics is theneed for such therapeutics to occupy a large number of receptors, whichis a formidable task since these receptors are widely expressed on allcells except red blood cells (Dinarello, Curr. Opin. Pharmacol.4:378-385, 2004). In most immune-mediated diseases, such as the diseasesdisclosed herein, the amount of IL-1β cytokine that is measurable inbody fluids or associated with activated cells is relatively low. Thus,a method of treatment and/or prevention that directly targets the IL-1βligand should provide a superior strategy, particularly whenadministering an IL-1β antibody with high affinity.

SUMMARY OF THE INVENTION

The present disclosure is directed to compositions and methods for thetreatment and/or prevention of gout in a subject. Such compositions andmethods may be used to treat a mammalian subject (e.g., human) sufferingfrom or at risk for gout. The methods and materials also may be used toprevent the occurrence of gout in an at risk subject.

In one aspect of the disclosure, a method of treating gout in a subject(e.g., human subject) is provided, the method comprising administering(e.g., in a therapeutically effective amount) an anti-IL-1β antibody orfragment thereof to the subject. In one embodiment of the disclosure,the gout is chronic gout. In another embodiment, the gout is acute gout.In yet another embodiment, the gout is refractory gout.

In another aspect, the disclosure provides a method of treating gout ina subject (e.g., human subject), the method comprising administering(e.g., in a therapeutically effective amount) an anti-IL-1β antibody orfragment thereof to the subject, wherein the dose of the antibody orfragment is sufficient to achieve at least a 50% reduction in jointpain. In one embodiment, the anti-IL-1β antibody or fragment thereof issufficient to achieve at least a 60% reduction in joint pain, at least a70% reduction in joint pain, at least a 80% reduction in joint pain, atleast a 90% reduction in joint pain, at least a 95% reduction in jointpain or a 100% reduction in joint pain.

In another aspect of the disclosure, the dose of the antibody orfragment is sufficient to achieve at least a 20% decrease in C-reactiveprotein (CRP) levels, at least a 30% decrease in CRP levels, at least a40% decrease in CRP levels, at least a 50% decrease in CRP levels, atleast a 60% decrease in CRP levels, at least a 70% decrease in CRPlevels, at least a 80% decrease in CRP levels, at least a 90% decreasein CRP levels. In a preferrred embodiment, the dose of the antibody orfragment is sufficient to achieve at least a 50% reduction in joint painand at least a 20% decrease in CRP levels, at least a 30% decrease inCRP levels, at least a 40% decrease in CRP levels, at least a 50%decrease in CRP levels, at least a 60% decrease in CRP levels, at leasta 70% decrease in CRP levels, at least a 80% decrease in CRP levels,and/or at least a 90% decrease in CRP levels.

In another aspect of the disclosure, the dose of the antibody orfragment is sufficient to achieve at least a 20% decrease in ErythrocyteSedimentation Rate (ESR), at least a 40% decrease in ESR, at least a 50%decrease in ESR, at least a 60% decrease in ESR, at least a 70% decreasein ESR, at least a 80% decrease in ESR, at least a 90% decrease in ESR.In a preferred embodiment, the dose of the antibody or fragment issufficient to achieve at least a 50% reduction in joint pain and atleast a 20% decrease in ESR, at least a 40% decrease in ESR, at least a50% decrease in ESR, at least a 60% decrease in ESR, at least a 70%decrease in ESR, at least a 80% decrease in ESR, and/or at least a 90%decrease in ESR

In another aspect, the disclosure provides a method of treating gout ina subject (e.g., human subject), the method comprising administering(e.g., in a therapeutically effective amount) an anti-IL-1β antibody orfragment thereof to the subject, wherein the dose of the antibody orfragment is sufficient to achieve at least a 50% reduction in jointpain, at least a 20% decrease in CRP levels and at least a 20% decreasein ESR. In one embodiment, the dose of the antibody or fragment issufficient to achieve at least a 50% reduction in joint pain, at least a30% decrease in CRP levels and a 30% decrease in ESR. In anotherembodiment, the dose of the antibody or fragment is sufficient toachieve at least a 50% reduction in joint pain, at least a 40% decreasein CRP levels and a 40% decrease in ESR. In another embodiment, the doseof the anti-IL-1β antibody or fragment is sufficient to achieve at leasta 60% reduction in joint pain, at least a 20% decrease in CRP levels andat least a 20% decrease in ESR. In another embodiment, the dose of theanti-IL-1β antibody or fragment is sufficient to achieve at least a 60%reduction in joint pain, at least a 40% decrease in CRP levels and atleast a 40% decrease in ESR. In another embodiment, the dose of theanti-IL-1β antibody or fragment is sufficient to achieve at least a 60%reduction in joint pain, at least a 50% decrease in CRP levels and atleast a 50% decrease in ESR. In yet another embodiment, the dose of theanti-IL-1β antibody or fragment is sufficient to achieve at least a 70%reduction in joint pain, at least a 20% decrease in CRP levels and atleast a 20% decrease in ESR. In another embodiment, the dose of theanti-IL-1β antibody or fragment is sufficient to achieve at least a 70%reduction in joint pain, at least a 40% decrease in CRP levels and atleast a 40% decrease in ESR. In another embodiment, the dose of theanti-IL-1β antibody or fragment is sufficient to achieve at least a 70%reduction in joint pain, at least a 50% decrease in CRP levels and atleast a 50% decrease in ESR.

The anti-IL-1β antibodies or antibody fragments used in the methods ofthe disclosed herein generally bind to IL-1β with high affinity. In onepreferred embodiment, the disclosure provides a method of treating goutin a subject (e.g., human subject), the method comprising administering(e.g., in a therapeutically effective amount) an anti-IL-1β antibody orfragment thereof to the subject, wherein, the antibody or antibodyfragment binds to IL-1β with a dissociation constant of about 10 nM orless, about 5 nM or less, about 1 nM or less, about 500 pM or less,about 250 pM or less, about 100 pM or less, about 50 pM or less, orabout 25 pM or less. In particularly preferred embodiments, the antibodyor antibody fragment binds to human IL-1β with a dissociation constantof about 100 pM or less, about 50 pM or less, about 10 pM or less, about5 pM or less, about 3 pM or less, about 1 pM or less, about 0.75 pM orless, about 0.5 pM or less, about 0.3 pM or less, about 0.2 pM or less,or about 0.1 pM or less.

In another aspect of the invention, the anti-IL-1β antibody or antibodyfragment is a neutralizing antibody. In another aspect, the anti-IL-1βantibody or antibody fragment binds to an IL-1β epitope such that thebound antibody or fragment substantially permits the binding of IL-1β toIL-1 receptor I (IL-1RI). In another aspect, the anti-IL-1β antibody orantibody fragment binds to IL-1β, but does not substantially prevent thebound IL-1β from binding to IL-1 receptor I (IL-1RI). In another aspect,the antibody or antibody fragment does not detectably bind to IL-1α,IL-1R or IL-1Ra. In yet another aspect of the invention, the antibody orantibody fragment binds to an epitope contained in the sequenceESVDPKNYPKKKMEKRFVFNKIE (SEQ ID NO: 1). In another aspect, the antibodyor fragment thereof competes with the binding of an antibody having thelight chain variable region of SEQ ID NO:5 and the heavy chain variableregion of SEQ ID NO:6

In yet another aspect of the invention, the antibody or antibodyfragment binds to an epitope incorporating Glu64 of IL-1β. In yetanother aspect of the invention, the antibody or antibody fragment bindsto amino acids 1-34 of the N terminus of IL-1β. Preferably, the antibodyor antibody fragment is human engineered, humanized or human.

In another aspect, the invention provides a method of treating a subject(e.g., mammal, human) displaying symptoms of, or at risk for, developinggout, the method comprising administering an anti-IL-1β antibody orfragment thereof to the subject in one or more doses.

In another aspect of the invention, a method is provided for treatinggout in a subject (e.g., mammal, human), the method comprisingadministering an anti-IL-1β antibody or fragment thereof to the human,wherein administration of an initial dose of the IL-1β antibody orantibody fragment is followed by the administration of one or moresubsequent doses. In one embodiment, administration of an initial doseof the antibody or antibody fragment is followed by the administrationof two or more subsequent doses. In another embodiment, administrationof an initial dose of the antibody or antibody fragment is followed bythe administration of one or more subsequent doses, and wherein said oneor more subsequent doses are in an amount that is approximately the sameor less than the initial dose. In another embodiment, administration ofan initial dose of the antibody or antibody fragment is followed by theadministration of one or more subsequent doses, and wherein at least oneof the subsequent doses is in an amount that is more than the initialdose. In yet another embodiment, administration of the antibody orantibody fragment is one time for each episode of acute gout. In theseembodiments, one may use, for example, an antibody or antibody fragment(e.g., a neutralizing antibody) which binds IL-1β with a dissociationconstant of less than 100 pM. Such an antibody or fragment thereof maycompete with the binding of an antibody having the light chain variableregion of SEQ ID NO:5 and the heavy chain variable region of SEQ ID NO:6to IL-1β.

In one embodiment, two or more, three or more, four or more, five ormore, six or more, seven or more, eight or more, nine or more, ten ormore or eleven or more subsequent doses of the antibody areadministered. In another embodiment administration of the initial doseand each one or more subsequent doses are separated from each other byan interval of at least about two weeks, at least about three weeks, atleast about one month, at least about two months, at least about threemonths, at least about four months, at least about five months, at leastabout six months, at least about seven months, at least about eightmonths, at least about nine months, at least about ten months, at leastabout eleven months, or at least about twelve months. In theseembodiments, one may use, for example, an antibody or antibody fragment(e.g., a neutralizing antibody) which binds IL-1β with a dissociationconstant of less than 100 pM. Such an antibody or fragment thereof maycompete with the binding of an antibody having the light chain variableregion of SEQ ID NO:5 and the heavy chain variable region of SEQ ID NO:6to IL-1β.

In another embodiment, the antibody or fragment is administered in oneor more doses of 5 mg/kg or less of antibody or fragment, 3 mg/kg orless of antibody or fragment, 2 mg/kg or less of antibody or fragment, 1mg/kg or less of antibody or fragment, 0.75 mg/kg or less of antibody orfragment, 0.5 mg/kg or less of antibody or fragment, 0.3 mg/kg or lessof antibody or fragment, 0.1 mg/kg or less of antibody or fragment, 0.03mg/kg or less of antibody or fragment, 0.01 mg/kg or less of antibody orfragment, 0.003 mg/kg or less of antibody or fragment or 0.001 mg/kg orless of antibody or fragment. Preferably, in each of the aforementionedembodiments, the antibody or fragment is administered in one or moredoses of at least 0.01 mg/kg of antibody or fragment, at least 0.01mg/kg of antibody or fragment, or at least 0.03 mg/kg of antibody orfragment. Preferably, the antibody or fragment is administered in one ormore doses of 0.001 mg/kg to 1 mg/kg, 0.001 mg/kg to 0.3 mg/kg, 0.003mg/kg to 1 mg/kg, 0.003 mg/kg to 0.3 mg/kg. The above dosage amountsrefer to mg (antibody or fragment)/kg (weight of the individual to betreated). In these embodiments, one may use, for example, an antibody orantibody fragment (e.g., a neutralizing antibody) which binds IL-1β witha dissociation constant of less than 100 pM. Such an antibody orfragment thereof may compete with the binding of an antibody having thelight chain variable region of SEQ ID NO:5 and the heavy chain variableregion of SEQ ID NO:6 to IL-1β.

In another embodiment, the initial dose and one or more subsequent dosesof antibody or fragment are each from about 0.01 mg/kg to about 10 mg/kgof antibody, from about 0.03 to about 1 mg/kg of antibody, from about0.03 to about 0.3 mg/kg of antibody, from about 0.05 to about 5 mg/kg ofantibody, from about 0.05 mg/kg to about 3 mg/kg of antibody, from about0.1 mg/kg to about 3 mg/kg of antibody, from about 0.1 mg/kg to about 1mg/kg of antibody, from about 0.1 mg/kg to about 0.5 mg/kg of antibody,from about 0.3 mg/kg to about 5 mg/kg of antibody, from about 0.3 mg/kgto about 3 mg/kg of antibody, from about 0.3 mg/kg to about 1 mg/kg ofantibody, from about 0.5 mg/kg to about 5 mg/kg of antibody, from about0.5 mg/kg to about 3 mg/kg of antibody, from about 0.5 mg/kg to about 1mg/kg of antibody, from about 1 mg/kg to about 5 mg/kg of antibody, orfrom about 1 mg/kg to about 3 mg/kg of antibody. In certain embodiments,two or more, three or more, four or more, five or more, six or more,seven or more, eight or more, nine or more, ten or more or eleven ormore subsequent doses of the antibody are administered. The above dosageamounts refer to mg (antibody or fragment)/kg (weight of the individualto be treated). The same applies hereinafter if a dosage amount ismentioned. In these embodiments, one may use, for example, an antibodyor antibody fragment (e.g., a neutralizing antibody) which binds IL-1βwith a dissociation constant of less than 100 pM. Such an antibody orfragment thereof may compete with the binding of an antibody having thelight chain variable region of SEQ ID NO:5 and the heavy chain variableregion of SEQ ID NO:6 to IL-1β.

In another aspect, the invention provides a method of treating gout in asubject (e.g., human), the method comprising administering atherapeutically effective amount of an anti-IL-1β antibody or fragmentthereof to the subject as an initial dose of about 5 mg/kg or less ofantibody or fragment, 3 mg/kg or less of antibody or fragment, 2 mg/kgor less of antibody or fragment, 1 mg/kg or less of antibody orfragment, 0.75 mg/kg or less of antibody or fragment, 0.5 mg/kg or lessof antibody or fragment, 0.3 mg/kg or less of antibody or fragment, 0.1mg/kg or less of antibody or fragment, or 0.03 mg/kg or less of antibodyor fragment, and a plurality of subsequent doses of antibody or fragmentin an amount about the same or less than the initial dose. In theseembodiments, one may use, for example, an antibody or antibody fragment(e.g., a neutralizing antibody) which binds IL-1β with a dissociationconstant of less than 100 pM. Such an antibody or fragment thereof maycompete with the binding of an antibody having the light chain variableregion of SEQ ID NO:5 and the heavy chain variable region of SEQ ID NO:6to IL-1β.

Preferably, in the aforementioned embodiments wherein the antibody orfragment is administered as an initial dose and a plurality ofsubsequent doses, the dose of antibody or fragment is at least 0.001mg/kg of antibody or fragment, at least 0.003 mg/kg of antibody orfragment, at least 0.01 mg/kg of antibody or fragment, at least, 0.03mg/kg of antibody or fragment, at least 0.05 mg/kg of antibody orfragment, or at least 0.09 mg/kg of antibody or fragment. In theseembodiments, one may use, for example, an antibody or antibody fragment(e.g., a neutralizing antibody) which binds IL-1β with a dissociationconstant of less than 100 pM. Such an antibody or fragment thereof maycompete with the binding of an antibody having the light chain variableregion of SEQ ID NO:5 and the heavy chain variable region of SEQ ID NO:6to IL-1β.

In yet another aspect of the invention, the antibody or fragment isadministered as a fixed dose, independent of a dose per subject weightratio. In one embodiment, the antibody or fragment is administered inone or more fixed doses of 1000 mg or less of antibody or fragment, 750mg or less of antibody or fragment, 500 mg or less of antibody orfragment, 250 mg or less of antibody or fragment, 100 mg or less ofantibody or fragment, about 25 mg or less of antibody or fragment, about10 mg or less of antibody or fragment or about 1.0 mg or less ofantibody or fragment. In another embodiment, the antibody or fragment isadministered in one or more fixed doses of at least about 0.1 mg ofantibody or fragment, at least about 1 mg of antibody or fragment, atleast about 5 mg of antibody or fragment, or at least about 10 mg ofantibody or fragment. In these embodiments, one may use, for example, anantibody or antibody fragment (e.g., a neutralizing antibody) whichbinds IL-1β with a dissociation constant of less than 100 pM. Such anantibody or fragment thereof may compete with the binding of an antibodyhaving the light chain variable region of SEQ ID NO:5 and the heavychain variable region of SEQ ID NO:6 to IL-1β.

In certain embodiments, the fixed dose is from about 1 mg to about 10mg, about 1 mg to about 25 mg, about 10 mg to about 25 mg, about 10 mgto about 50 mg, about 10 mg to about 100 mg, about 25 mg to about 50 mg,about 25 mg to about 100 mg, about 50 mg to about 100 mg, about 50 mg toabout 150 mg, about 100 mg to about 150 mg, about 100 mg to about 200mg, about 150 mg to about 200 mg, about 150 mg to about 250 mg, about200 mg to about 250 mg, about 200 mg to about 300 mg, about 250 mg toabout 300 mg, about 250 mg to about 500 mg, about 300 mg to about 400mg, about 400 mg to about 500 mg, about 400 mg to about 600 mg, about500 mg to about 750 mg, about 600 mg to about 750 mg, about 700 mg toabout 800 mg, about 750 mg to about 1000 mg. In a preferred embodiment,the fixed dose is administered in one or more doses of about 0.1 mg toabout 100 mg, about 1.0 mg to about 100 mg or about 1.0 mg to about 50mg. In another preferred embodiment, the fixed dose is selected from thegroup consisting of about 1 mg to about 10 mg, about 1 mg to about 25mg, about 10 mg to about 25 mg, about 10 mg to about 100 mg, about 25 mgto about 50 mg, about 50 mg to about 100 mg, about 100 mg to about 150mg, about 150 mg to about 200 mg, about 200 mg to about 250 mg. In theseembodiments, one may use, for example, an antibody or antibody fragment(e.g., a neutralizing antibody) which binds IL-1β with a dissociationconstant of less than 100 pM. Such an antibody or fragment thereof maycompete with the binding of an antibody having the light chain variableregion of SEQ ID NO:5 and the heavy chain variable region of SEQ ID NO:6to IL-1β.

In another aspect, the invention provides a method of treating gout in asubject, the method comprising administering a therapeutically effectiveamount of an anti-IL-1β antibody or fragment thereof to the subject,wherein administration of an initial dose of the antibody or antibodyfragment is followed by the administration of one or more subsequentdoses, and wherein the plasma concentration of said antibody or antibodyfragment in the human is permitted to decrease below a level of about0.1 ug/mL for a period of time greater than about 1 week and less thanabout 6 months between administrations during a course of treatment withsaid initial dose and one or more subsequent doses. In one embodiment,the plasma concentration of said antibody or antibody fragment ispermitted to decrease below a level of about 0.07 ug/mL, about 0.05ug/mL, about 0.03 ug/mL or about 0.01 ug/mL for a period of time greaterthan about 1 week and less than about 5 months, about 4 months, about 3months, about 2 months, about 1 month, about 3 weeks, or about 2 weeksbetween administrations. In one embodiment, these plasma values refer tovalues obtained for an individual that is treated with the antibody offragment in accordance with the invention. In one embodiment, such anindividual may be a patient suffering from gout. In these embodiments,one may use, for example, an antibody or antibody fragment (e.g., aneutralizing antibody) which binds IL-1β with a dissociation constant ofless than 100 pM. Such an antibody or fragment thereof may compete withthe binding of an antibody having the light chain variable region of SEQID NO:5 and the heavy chain variable region of SEQ ID NO:6 to IL-1β.

The invention contemplates that an anti-IL-1β antibody or fragment usedin accordance with the methods herein may be administered in any of theaforementioned dose amounts, numbers of subsequent administrations, anddosing intervals between administrations, and that any of the discloseddose amounts, numbers of subsequent administrations, and dosingintervals between administrations may be combined with each other inalternative regimens to modulate the therapeutic benefit. In certainembodiments, the one or more subsequent doses are in an amount that isapproximately the same or less than the first dose administered. Inanother embodiment, the one or more subsequent doses are in an amountthat is approximately more than the first dose administered. Preferablythe anti-IL-1β antibody or fragment is administered by subcutaneous,intramuscular or intravenous injection. The invention contemplates thateach dose of antibody or fragment may be administered at one or moresites. In these embodiments, one may use, for example, an antibody orantibody fragment (e.g., a neutralizing antibody) which binds IL-1β witha dissociation constant of less than 100 pM. Such an antibody orfragment thereof may compete with the binding of an antibody having thelight chain variable region of SEQ ID NO:5 and the heavy chain variableregion of SEQ ID NO:6 to IL-1β.

In one embodiment, the anti-IL-1β antibody or fragment is administeredin combination with at least one other medically accepted treatment forthe disease, condition or complication. In another embodiment, the atleast one other medically accepted treatment for the disease, conditionor complication is reduced or discontinued, while treatment with theanti-IL-1β antibody or fragment is maintained at a constant dosingregimen. In another embodiment, the at least one other medicallyaccepted treatment for the disease, condition or complication is reducedor discontinued, and treatment with the anti-IL-1β antibody or fragmentis reduced. In another embodiment, the at least one other medicallyaccepted treatment for the disease, condition or complication is reducedor discontinued, and treatment with the anti-IL-1β antibody or fragmentis increased. In yet another embodiment, the at least one othermedically accepted treatment for the disease, condition or complicationis maintained and treatment with the anti-IL-1β antibody or fragment isreduced or discontinued. In yet another embodiment, the at least oneother medically accepted treatment for the disease, condition orcomplication and treatment with the anti-IL-1β antibody or fragment arereduced or discontinued. In these embodiments, one may use, for example,an antibody or antibody fragment (e.g., a neutralizing antibody) whichbinds IL-1β with a dissociation constant of less than 100 pM. Such anantibody or fragment thereof may compete with the binding of an antibodyhaving the light chain variable region of SEQ ID NO:5 and the heavychain variable region of SEQ ID NO:6 to IL-1β.

In another aspect, methods provided herein are in conjunction with atleast one additional treatment method, said additional treatment methodcomprising administering at least one pharmaceutical compositioncomprising an active agent other than an IL-1β antibody or fragment. Inyet another aspect, the methods prevent or delay the need for at leastone additional treatment method, said additional treatment methodcomprising administering at least one pharmaceutical compositioncomprising an active agent other than an IL-1β antibody or fragment. Instill another aspect, the methods reduce the amount, frequency orduration of at least one additional treatment method, said additionaltreatment method comprising administering at least one pharmaceuticalcomposition comprising an active agent other than an IL-1β antibody orfragment. In yet another embodiment, treatment with the at least oneactive agent is maintained. In another embodiment, treatment with the atleast one active agent is reduced or discontinued, while treatment withthe anti-IL-1β antibody or fragment is maintained at a constant dosingregimen. In another embodiment, treatment with the at least one activeagent is reduced or discontinued and treatment with the anti-IL-1βantibody or fragment is reduced. In another embodiment, treatment withthe at least one active agent is is reduced or discontinued, andtreatment with the anti-IL-1β antibody or fragment is increased. In yetanother embodiment, treatment with the at least one active agent ismaintained and treatment with the anti-IL-1β antibody or fragment isreduced or discontinued. In yet another embodiment, treatment with theat least one active agent and treatment with the anti-IL-1β antibody orfragment are reduced or discontinued. In these embodiments, one may use,for example, an antibody or antibody fragment (e.g., a neutralizingantibody) which binds IL-1β with a dissociation constant of less than100 pM. Such an antibody or fragment thereof may compete with thebinding of an antibody having the light chain variable region of SEQ IDNO:5 and the heavy chain variable region of SEQ ID NO:6 to IL-1β.

In another aspect, the invention provides a method of treating gout in asubject, the method comprising administering a therapeutically effectiveamount of an anti-IL-1β antibody or fragment thereof to the subject,wherein administration of an initial dose of the antibody or antibodyfragment is followed by the administration of one or more subsequentdoses, and wherein the plasma concentration of said antibody or antibodyfragment in the human is maintained at a level of at least about 0.03ug/mL, at least about 0.05 ug/mL, at least about 0.08 ug/mL, at leastabout 0.1 ug/mL, at least about 0.15 ug/mL, at least about 0.2 ug/mL, atleast about 0.25 ug/mL, at least about 0.3 ug/mL, at least about 0.4ug/mL, at least about 0.5 ug/mL, at least about 0.6 ug/mL, at leastabout 0.8 ug/mL, at least about 1 ug/mL, at least about 1.5 ug/mL, atleast about 2 ug/mL, at least about 3 ug/mL, at least about 4 ug/mL, orat least about 5 ug/mL during a course of treatment with said initialdose and one or more subsequent doses. In one embodiment, these plasmavalues refer to values obtained for an individual that is treated withthe antibody of fragment in accordance with the invention. In oneembodiment, such an individual may be a patient suffering from gout. Inthese embodiments, one may use, for example, an antibody or antibodyfragment (e.g., a neutralizing antibody) which binds IL-1β with adissociation constant of less than 100 pM. Such an antibody or fragmentthereof may compete with the binding of an antibody having the lightchain variable region of SEQ ID NO:5 and the heavy chain variable regionof SEQ ID NO:6 to IL-1β.

In another aspect, the invention provides a method of treating gout in asubject, the method comprising administering a therapeutically effectiveamount of an anti-IL-1β antibody or fragment thereof to the subject,wherein the antibody or fragment thereof has a lower IC₅₀ than an IL-1βreceptor antagonist in a human whole blood IL-1β inhibition assay thatmeasures IL-1β induced production of IL-8. In one embodiment, theantibody or fragment has an IC₅₀ that is less than about 90%, 80%, 70%,60%, 50% of the IC₅₀ of an IL-1β receptor antagonist in a human wholeblood IL-1β inhibition assay that measures IL-1β induced production ofIL-8. In a further embodiment, the antibody or fragment has an IC₅₀ thatis less than about 40%, 30%, 20%, 10% of the IC₅₀ of an IL-1β receptorantagonist in a human whole blood IL-1β inhibition assay that measuresIL-1β induced production of IL-8. In a preferred embodiment, theantibody or fragment has an IC₅₀ that is less than about 8%, 5%, 4%, 3%,2%, 1% of the IC₅₀ of an IL-1β receptor antagonist in a human wholeblood IL-1β inhibition assay that measures IL-1β induced production ofIL-8. In one embodiment, the IL-1β receptor antagonist is anakinra(i.e., Kineret®). In these embodiments, one may use, for example, anantibody or antibody fragment (e.g., a neutralizing antibody) whichbinds IL-1β with a dissociation constant of less than 100 pM. Such anantibody or fragment thereof may compete with the binding of an antibodyhaving the light chain variable region of SEQ ID NO:5 and the heavychain variable region of SEQ ID NO:6 to IL-1β.

In another aspect, the invention provides a method of treating gout in asubject, the method comprising administering a therapeutically effectiveamount of an anti-IL-1β antibody or fragment thereof to the subject,wherein the antibody or fragment thereof provides in vivo inhibition ofIL-11 stimulated release of IL-6 in mice compared to a control antibodyusing an assay that is described by Economides et al., Nature Med.,9:47-52 (2003) which is incorporated by reference. In one embodiment theantibody or fragment provides in vivo inhibition of IL-1ß stimulatedrelease of IL-6 in mice of at least about 10%, 20%, 30%, 40%, 50%compared to the control antibody. In a further embodiment, the antibodyor fragment provides in vivo inhibition of IL-1ß stimulated release ofIL-6 in mice of at least about 60%, 70%, 80%, 90%, 95% compared to thecontrol antibody. In one embodiment, the control antibody is an isotypecontrol antibody. In these embodiments, one may use, for example, anantibody or antibody fragment (e.g., a neutralizing antibody) whichbinds IL-1β with a dissociation constant of less than 100 pM. Such anantibody or fragment thereof may compete with the binding of an antibodyhaving the light chain variable region of SEQ ID NO:5 and the heavychain variable region of SEQ ID NO:6 to IL-1β.

In another aspect, the invention provides a method of treating gout in asubject, the method comprising administering a therapeutically effectiveamount of an anti-IL-1β antibody or fragment thereof to the subject,wherein the antibody or fragment thereof inhibits Staphylococcusepidermidis induced cytokine production in human whole blood compared toa control where no antibody is used. In one embodiment the antibody orfragment provides a greater level of inhibition of Staphylococcusepidermidis induced cytokine production in human whole blood by at leastabout 10%, 20%, 30%, 40%, 50% compared to the control. In a furtherembodiment, the antibody or fragment provides a greater level ofinhibition of Staphylococcus epidermidis induced cytokine production inhuman whole blood by at least about 60%, 70%, 80%, 90%, 95% compared tothe control. In one embodiment, the inhibited cytokines are IL-1ß,IL-1α, IL-6, IL-8, IL-1Ra, TNFα or IFNγ. In these embodiments, one mayuse, for example, an antibody or antibody fragment (e.g., a neutralizingantibody) which binds IL-1β with a dissociation constant of less than100 pM. Such an antibody or fragment thereof may compete with thebinding of an antibody having the light chain variable region of SEQ IDNO:5 and the heavy chain variable region of SEQ ID NO:6 to IL-1β.

In another aspect, the invention discloses the use of an anti-IL-1βantibody or fragment thereof which as a lower IC₅₀ than an IL-1βreceptor antagonist in a human whole blood IL-1β inhibition assay thatmeasures IL-1β induced production of IL-8, in the manufacture of acomposition for use in the treatment of gout. In one embodiment, theIL-1β receptor antagonist is anakinra (i.e., Kineret®). In theseembodiments, one may use, for example, an antibody or antibody fragment(e.g., a neutralizing antibody) which binds IL-1β with a dissociationconstant of less than 100 pM. Such an antibody or fragment thereof maycompete with the binding of an antibody having the light chain variableregion of SEQ ID NO:5 and the heavy chain variable region of SEQ ID NO:6to IL-1β.

In another aspect of the invention, the use of the IL-1β antibodies orbinding fragments is contemplated in the manufacture of a medicament fortreating or preventing a disease or condition as disclosed herein. Inany of the uses, the medicament can be coordinated with treatment usinga second active agent. In another embodiment of the invention, the useof a synergistic combination of an antibody of the invention forpreparation of a medicament for treating a patient exhibiting symptomsof at risk for developing a disease or condition as disclosed herein,wherein the medicament is coordinated with treatment using a secondactive agent is contemplated. Embodiments of any of the aforementioneduses are contemplated wherein the amount of the IL-1β binding antibodyor fragment in the medicament is at a dose effective to reduce thedosage of second active agent required to achieve a therapeutic effect.In these embodiments, one may use, for example, an antibody or antibodyfragment (e.g., a neutralizing antibody) which binds IL-1β with adissociation constant of less than 100 pM. Such an antibody or fragmentthereof may compete with the binding of an antibody having the lightchain variable region of SEQ ID NO:5 and the heavy chain variable regionof SEQ ID NO:6 to IL-1β.

In yet another aspect of the invention, an article of manufacture isprovided, comprising a container, a composition within the containercomprising an anti-IL-1β antibody or fragment thereof, and a packageinsert containing instructions to administer the antibody or fragment toa human in need of treatment according to the aforementioned methods ofthe invention. In one embodiment, the container further comprises apharmaceutically suitable carrier, excipient or diluent. In a relatedembodiment, the composition within the container further comprises asecond active agent. In these embodiments, one may use, for example, anantibody or antibody fragment (e.g., a neutralizing antibody) whichbinds IL-1β with a dissociation constant of less than 100 pM. Such anantibody or fragment thereof may compete with the binding of an antibodyhaving the light chain variable region of SEQ ID NO:5 and the heavychain variable region of SEQ ID NO:6 to IL-1β.

Kits are also contemplated by the present invention. In one embodiment,a kit comprises a therapeutically or prophylactically effective amountof an anti-IL-1β antibody or fragment, packaged in a container, such asa vial or bottle, and further comprising a label attached to or packagedwith the container, the label describing the contents of the containerand providing indications and/or instructions regarding use of thecontents of the container for treatment or prevention of a disease orcondition according to the aforementioned methods of the invention. Inone embodiment, the container further comprises a pharmaceuticallysuitable carrier, excipient or diluent. In a related embodiment, thecontainer further contains a second active agent. In these embodiments,one may use, for example, an antibody or antibody fragment (e.g., aneutralizing antibody) which binds IL-1β with a dissociation constant ofless than 100 pM. Such an antibody or fragment thereof may compete withthe binding of an antibody having the light chain variable region of SEQID NO:5 and the heavy chain variable region of SEQ ID NO:6 to IL-1β.

In one embodiment, the article of manufacture, kit or medicament is forthe treatment or prevention of gout in a subject. In another embodiment,the instructions of a package insert of an article of manufacture orlabel of a kit comprise instructions for administration of the antibodyor fragment according to any of the aforementioned dose amounts, numbersof subsequent administrations, and dosing intervals betweenadministrations, as well as any combination of dose amounts numbers ofsubsequent administrations, and dosing intervals between administrationsdescribed herein. In yet another embodiment, the container of kit orarticle of manufacture is a pre-filled syringe. In these embodiments,one may use, for example, an antibody or antibody fragment (e.g., aneutralizing antibody) which binds IL-1β with a dissociation constant ofless than 100 pM. Such an antibody or fragment thereof may compete withthe binding of an antibody having the light chain variable region of SEQID NO:5 and the heavy chain variable region of SEQ ID NO:6 to IL-1β.

In another aspect of the disclosure, a method of treating monosodiumurate (MSU) crystal-induced release of a pro-inflammatory cytokine in asubject (e.g., human subject) is provided, the method comprisingadministering a therapeutically effective amount of an anti-IL-1βantibody or fragment thereof to the subject. In one embodiment, thepro-inflammatory cytokine is IL-1β. In another embodiment, thepro-inflammatory cytokine is IL-6. In these embodiments, one may use,for example, an antibody or antibody fragment (e.g., a neutralizingantibody) which binds IL-1β with a dissociation constant of less than100 pM. Such an antibody or fragment thereof may compete with thebinding of an antibody having the light chain variable region of SEQ IDNO:5 and the heavy chain variable region of SEQ ID NO:6 to IL-1β.

It is to be understood that where the present specification mentionsmethods of treatments making use of antibodies or fragments thereof withcertain properties (such as Kd values or IC₅₀ values), this also meansto embody the use of such antibodies or fragments thereof in themanufacture of a medicament for use in these methods. Further, theinvention also encompasses antibodies or fragments thereof having theseproperties as well as pharmaceutical compositions comprising theseantibodies or fragments thereof for use in the methods of treatmentdiscussed hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the results of an in vitro IL-1β inhibitionexperiment for the antibody designated AB7 and for Kineret® involvingIL-1 induced production of IL-8.

FIG. 2A is a graph showing the results of an in vivo IL-1β inhibitionexperiment for the antibodies designated AB5 and AB7 involving IL-1stimulated release of IL-6.

FIG. 2B is a graph showing the results of an in vivo IL-1β inhibitionexperiment for the antibodies designated AB7 involving IL-1 stimulatedrelease of IL-6, and comparing inhibition of human (panel A) versusmouse (panel B) IL-1β.

FIG. 3 is a graph showing serum concentrations following administration0.1, 1 or 10 mg/kg of an anti-IL-1β antibody in rats.

FIG. 4 is a graph showing serum concentrations following administrationof 0.3 or 3 mg/kg of an anti-IL-1β antibody in Cynomolgus monkeys.

FIG. 5 is a graph modeling plasma concentration profiles of ananti-IL-1β antibody in Cynomolgus monkeys following five monthly dosesof 0.1, 0.3, 1 or 3 mg/kg.

FIG. 6 is a table showing reduction of Staphyloccus epidermidis-inducedcytokine production in human whole blood by treatment with an anti-IL-1βantibody.

FIG. 7 is a graph showing the pharmacokinetics of AB7 in humansfollowing administration of a dose of 0.01 mg/kg of antibody.

FIG. 8 is a graph showing serum concentrations following administrationof 0.01, 0.03, 0.1, 0.3, or 1.0 mg/kg of an anti-IL-1β antibody in humansubjects with Type 2 diabetes.

FIG. 9 is a graph showing median percent change in CRP at day 28following administration of 0.01, 0.03, 0.1, 0.3, or 1.0 mg/kg of ananti-IL-1β antibody to human subjects with Type 2 diabetes.

FIGS. 10A and 10B are graphs showing efficacy of an anti-IL-1β antibodyin a mouse model of MSU-crystal induced acute gout.

DETAILED DESCRIPTION

The present disclosure is directed to methods and related articles ofmanufacture for the treatment of gout (e.g., acute gout, chronic gout,refractory gout) in a subject, the method comprising administering tothe subject one or more doses of an anti-IL-1β antibody or fragmentthereof. Because of the problems with current treatments, new therapiesto treat gout are needed to replace or complement availablepharmaceutical approaches. The methods disclosed herein comprise, forexample, administering an anti-IL-1β antibody or fragment thereof.Methods that directly target the IL-1β ligand with an antibody,particularly antibodies that exhibit high affinity, provide advantagesover other potential methods of treatment, such as IL-1β receptorantagonists (e.g., IL-1Ra, Anakinra, Kineret®). The challenge for IL-1receptor antagonist-based therapeutics is the need for such therapeuticsto occupy a large number of receptors, which is a formidable task sincethese receptors are widely expressed on all cells except red blood cells(Dinarello, Curr. Opin. Pharmacol. 4:378-385, 2004). In mostimmune-mediated diseases, such as the diseases disclosed herein, theamount of IL-1β cytokine that is measurable in body fluids or associatedwith activated cells is relatively low. As illustrated in Examplesbelow, we have surprisingly found that antibodies, such as thosedisclosed herein, can be used to achieve the desired level of activityover a broad range of doses, including at very low doses. Thus, a methodof treatment and/or prevention that directly targets the IL-1β ligandshould provide a superior strategy.

IL-1β is a pro-inflammatory cytokine secreted by a number of differentcell types including monocytes and macrophages. When released as part ofan inflammatory reaction, IL-1β produces a range of biological effects,mainly mediated through induction of other inflammatory mediators suchas corticotrophin, platelet factor-4, prostaglandin E2 (PGE2), IL-6, andIL-8. IL-1β induces both local and systemic inflammatory effects throughthe activation of the IL-1 receptor found on almost all cell types.

The interleukin-1 (IL-1) family of cytokines has been implicated inseveral disease states such as rheumatoid arthritis (RA),osteoarthritis, Crohn's disease, ulcerative colitis (UC), septic shock,chronic obstructive pulmonary disease (COPD), asthma, graft versus hostdisease, atherosclerosis, adult T-cell leukemia, multiple myeloma,multiple sclerosis, stroke, and Alzheimer's disease. IL-1 family membersinclude IL-1α, IL-1β, and IL-1Ra. Although related by their ability tobind to IL-1 receptors (IL-1R1, IL-1R2), each of these cytokines isexpressed by a different gene and has a different primary amino acidsequence. Furthermore, the physiological activities of these cytokinescan be distinguished from each other.

Compounds that disrupt IL-1 receptor signaling have been investigated astherapeutic agents to treat IL-1 mediated diseases, such as for examplesome of the aforementioned diseases. These compounds include recombinantIL-1Ra (Amgen Inc., Thousand Oaks, Calif.), IL-1 receptor “trap” peptide(Regeneron Inc., Tarrytown, N.Y.), as well as animal-derived IL-1βantibodies and recombinant IL-1β antibodies and fragments thereof.

As noted above, IL-1 receptor antagonist (IL-1Ra) polypeptide has beensuggested for use in the treatment of gout (So et al., 2007, ibid;McGonagle et al., 2007, ibid), but there remains a need for effectivemeans to treat gout, particularly those that do not require daily,repeated injections. An additional challenge for IL-1 receptorantagonist-based therapeutics is the need for such therapeutics tooccupy a large number of receptors, which is a formidable task sincethese receptors are widely expressed on all cells except red blood cells(Dinarello, Curr. Opin. Pharmacol. 4:378-385, 2004). In mostimmune-mediated diseases, such as the diseases disclosed herein, theamount of IL-1β cytokine that is measurable in body fluids or associatedwith activated cells is relatively low. Thus, a method of treatmentand/or prevention that directly targets the IL-1β ligand is a superiorstrategy, particularly when administering an IL-1β antibody with highaffinity.

The present invention provides methods and related compositions andarticles of manufacture for the treatment and/or prevention of gout in asubject (e.g., mammalian, human), using an antibody or fragment thereofspecific for IL-1β.

As shown in Example 1 below, we have surprisingly found that such anantibody (e.g., with very high affinity) can be far more potent aninhibitor of the IL-1 pathway than is IL-Ra (e.g., Kineret®), andprovides an opportunity to achieve a therapeutic effect at a lower doseand/or with less frequent administration than necessary for other drugs,such as recombinant IL-1Ra.

Such methods as described herein with an IL-1β antibody or fragment mayinclude the treatment of a subject suffering from gout (e.g., acutegout, chronic gout, refractory gout). The methods also may includepreventing the occurrence of gout (e.g., acute gout, chronic gout,refractory gout) in an at risk subject.

Antibodies, Humanized Antibodies, and Human Engineered Antibodies

The IL-1 (e.g., IL-1β) binding antibodies of the present invention maybe provided as polyclonal antibodies, monoclonal antibodies (mAbs),recombinant antibodies, chimeric antibodies, CDR-grafted antibodies,fully human antibodies, single chain antibodies, and/or bispecificantibodies, as well as fragments, including variants and derivativesthereof, provided by known techniques, including, but not limited toenzymatic cleavage, peptide synthesis or recombinant techniques.

Antibodies generally comprise two heavy chain polypeptides and two lightchain polypeptides, though single domain antibodies having one heavychain and one light chain, and heavy chain antibodies devoid of lightchains are also contemplated. There are five types of heavy chains,called alpha, delta, epsilon, gamma and mu, based on the amino acidsequence of the heavy chain constant domain. These different types ofheavy chains give rise to five classes of antibodies, IgA (includingIgA₁ and IgA₂), IgD, IgE, IgG and IgM, respectively, including foursubclasses of IgG, namely IgG₁, IgG₂, IgG₃ and IgG₄. There are also twotypes of light chains, called kappa (κ) or lambda (A) based on the aminoacid sequence of the constant domains. A full-length antibody includes aconstant domain and a variable domain. The constant region need not bepresent in an antigen binding fragment of an antibody. Antigen bindingfragments of an antibody disclosed herein can include Fab, Fab′,F(ab′)₂, and F(v) antibody fragments. As discussed in more detail below,IL-1β binding fragments encompass antibody fragments and antigen-bindingpolypeptides that will bind IL-1β.

Each of the heavy chain and light chain sequences of an antibody, orantigen binding fragment thereof, includes a variable region with threecomplementarity determining regions (CDRs) as well as non-CDR frameworkregions (FRs). The terms “heavy chain” and “light chain,” as usedherein, mean the heavy chain variable region and the light chainvariable region, respectively, unless otherwise noted. Heavy chain CDRsare referred to herein as CDR-H1, CDR-H2, and CDR-H3. Light chain CDRsare referred to herein as CDR-L1, CDR-L2, and CDR-L3. Variable regionsand CDRs in an antibody sequence can be identified (i) according togeneral rules that have been developed in the art or (ii) by aligningthe sequences against a database of known variable regions. Methods foridentifying these regions are described in Kontermann and Dubel, eds.,Antibody Engineering, Springer, New York, N.Y., 2001, and Dinarello etal., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken,N.J., 2000. Databases of antibody sequences are described in and can beaccessed through “The Kabatman” database at www.bioinf.org.uk/abs(maintained by A. C. Martin in the Department of Biochemistry &Molecular Biology University College London, London, England) and VBASE2at www.vbase2.org, as described in Retter et al., Nucl. Acids Res.,33(Database issue): D671-D674 (2005). The “Kabatman” database web sitealso includes general rules of thumb for identifying CDRs. The term“CDR,” as used herein, is as defined in Kabat et al., Sequences ofImmunological Interest, 5^(th) ed., U.S. Department of Health and HumanServices, 1991, unless otherwise indicated.

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. An improved antibody response may be obtainedby conjugating the relevant antigen to a protein that is immunogenic inthe species to be immunized, e.g., keyhole limpet hemocyanin, serumalbumin, bovine thyroglobulin, or soybean trypsin inhibitor using abifunctional or derivatizing agent, for example, maleimidobenzoylsulfosuccinimide ester (conjugation through cysteine residues),N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinicanhydride or other agents known in the art.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 pg or 5 pg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later, the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. At 7-14 days post-boosterinjection, the animals are bled and the serum is assayed for antibodytiter. Animals are boosted until the titer plateaus. Preferably, theanimal is boosted with the conjugate of the same antigen, but conjugatedto a different protein and/or through a different cross-linking reagent.Conjugates also can be made in recombinant cell culture as proteinfusions. Also, aggregating agents such as alum are suitably used toenhance the immune response.

Monoclonal antibody refers to an antibody obtained from a population ofsubstantially homogeneous antibodies. Monoclonal antibodies aregenerally highly specific, and may be directed against a singleantigenic site, in contrast to conventional (polyclonal) antibodypreparations that typically include different antibodies directedagainst different determinants (epitopes). In addition to theirspecificity, the monoclonal antibodies are advantageous in that they aresynthesized by the homogeneous culture, uncontaminated by otherimmunoglobulins with different specificities and characteristics.

Monoclonal antibodies to be used in accordance with the presentinvention may be made by the hybridoma method first described by Kohleret al., (Nature, 256:495-7, 1975), or may be made by recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567). The monoclonal antibodiesmay also be isolated from phage antibody libraries using the techniquesdescribed in, for example, Clackson et al., (Nature 352:624-628, 1991)and Marks et al., (J. Mol. Biol. 222:581-597, 1991).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster or macaque monkey, is immunized as herein described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofhuman monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).Exemplary murine myeloma lines include those derived from MOP-21 andM.C.-11 mouse tumors available from the Salk Institute Cell DistributionCenter, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells availablefrom the American Type Culture Collection, Rockville, Md. USA.

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). The binding affinity of the monoclonalantibody can, for example, be determined by Scatchard analysis (Munsonet al., Anal. Biochem., 107:220 (1980)).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, DMEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal. Themonoclonal antibodies secreted by the subclones are suitably separatedfrom the culture medium, ascites fluid, or serum by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

It is further contemplated that antibodies of the invention may be usedas smaller antigen binding fragments of the antibody well-known in theart and described herein.

The present invention encompasses IL-1 (e.g., IL-1β) binding antibodiesthat include two full length heavy chains and two full length lightchains. Alternatively, the IL-1β binding antibodies can be constructssuch as single chain antibodies or “mini” antibodies that retain bindingactivity to IL-1β. Such constructs can be prepared by methods known inthe art such as, for example, the PCR mediated cloning and assembly ofsingle chain antibodies for expression in E. coli (as described inAntibody Engineering, The practical approach series, J. McCafferty, H.R. Hoogenboom, and D. J. Chiswell, editors, Oxford University Press,1996). In this type of construct, the variable portions of the heavy andlight chains of an antibody molecule are PCR amplified from cDNA. Theresulting amplicons are then assembled, for example, in a second PCRstep, through a linker DNA that encodes a flexible protein linkercomposed of the amino acids Gly and Ser. This linker allows the variableheavy and light chain portions to fold in such a way that the antigenbinding pocket is regenerated and antigen is bound with affinities oftencomparable to the parent full-length dimeric immunoglobulin molecule.

The IL-1 (e.g., IL-1β) binding antibodies and fragments of the presentinvention encompass variants of the exemplary antibodies, fragments andsequences disclosed herein. Variants include peptides and polypeptidescomprising one or more amino acid sequence substitutions, deletions,and/or additions that have the same or substantially the same affinityand specificity of epitope binding as one or more of the exemplaryantibodies, fragments and sequences disclosed herein. Thus, variantsinclude peptides and polypeptides comprising one or more amino acidsequence substitutions, deletions, and/or additions to the exemplaryantibodies, fragments and sequences disclosed herein where suchsubstitutions, deletions and/or additions do not cause substantialchanges in affinity and specificity of epitope binding. For example, avariant of an antibody or fragment may result from one or more changesto an antibody or fragment, where the changed antibody or fragment hasthe same or substantially the same affinity and specificity of epitopebinding as the starting sequence. Variants may be naturally occurring,such as allelic or splice variants, or may be artificially constructed.Variants may be prepared from the corresponding nucleic acid moleculesencoding said variants. Variants of the present antibodies and IL-1βbinding fragments may have changes in light and/or heavy chain aminoacid sequences that are naturally occurring or are introduced by invitro engineering of native sequences using recombinant DNA techniques.Naturally occurring variants include “somatic” variants which aregenerated in vivo in the corresponding germ line nucleotide sequencesduring the generation of an antibody response to a foreign antigen.

Variants of IL-1 (e.g., IL-1β) binding antibodies and binding fragmentsmay also be prepared by mutagenesis techniques. For example, amino acidchanges may be introduced at random throughout an antibody coding regionand the resulting variants may be screened for binding affinity forIL-1β or for another property. Alternatively, amino acid changes may beintroduced in selected regions of an IL-1β antibody, such as in thelight and/or heavy chain CDRs, and/or in the framework regions, and theresulting antibodies may be screened for binding to IL-1β or some otheractivity. Amino acid changes encompass one or more amino acidsubstitutions in a CDR, ranging from a single amino acid difference tothe introduction of multiple permutations of amino acids within a givenCDR, such as CDR3. In another method, the contribution of each residuewithin a CDR to IL-1β binding may be assessed by substituting at leastone residue within the CDR with alanine. Lewis et al. (1995), Mol.Immunol. 32: 1065-72. Residues which are not optimal for binding toIL-1β may then be changed in order to determine a more optimum sequence.Also encompassed are variants generated by insertion of amino acids toincrease the size of a CDR, such as CDR3. For example, most light chainCDR3 sequences are nine amino acids in length. Light chain sequences inan antibody which are shorter than nine residues may be optimized forbinding to IL-1 β by insertion of appropriate amino acids to increasethe length of the CDR.

Variants may also be prepared by “chain shuffling” of light or heavychains. Marks et al. (1992), Biotechnology 10: 779-83. A single light(or heavy) chain can be combined with a library having a repertoire ofheavy (or light) chains and the resulting population is screened for adesired activity, such as binding to IL-1β. This permits screening of agreater sample of different heavy (or light) chains in combination witha single light (or heavy) chain than is possible with librariescomprising repertoires of both heavy and light chains.

The IL-1 (e.g., IL-1β) binding antibodies and fragments of the presentinvention encompass derivatives of the exemplary antibodies, fragmentsand sequences disclosed herein. Derivatives include polypeptides orpeptides, or variants, fragments or derivatives thereof, which have beenchemically modified. Examples include covalent attachment of one or morepolymers, such as water soluble polymers, N-linked, or O-linkedcarbohydrates, sugars, phosphates, and/or other such molecules. Thederivatives are modified in a manner that is different from naturallyoccurring or starting peptide or polypeptides, either in the type orlocation of the molecules attached. Derivatives further include deletionof one or more chemical groups which are naturally present on thepeptide or polypeptide.

The IL-1β binding antibodies and fragments of the present invention canbe bispecific. Bispecific antibodies or fragments can be of severalconfigurations. For example, bispecific antibodies may resemble singleantibodies (or antibody fragments) but have two different antigenbinding sites (variable regions). Bispecific antibodies can be producedby chemical techniques (Kranz et al. (1981), Proc. Natl. Acad. Sci. USA,78: 5807), by “polydoma” techniques (U.S. Pat. No. 4,474,893) or byrecombinant DNA techniques. Bispecific antibodies of the presentinvention can have binding specificities for at least two differentepitopes, at least one of which is an epitope of IL-1β. The IL-1βbinding antibodies and fragments can also be heteroantibodies.Heteroantibodies are two or more antibodies, or antibody bindingfragments (Fab) linked together, each antibody or fragment having adifferent specificity.

Techniques for creating recombinant DNA versions of the antigen-bindingregions of antibody molecules which bypass the generation of monoclonalantibodies are contemplated for the present IL-1 (e.g., IL-1β) bindingantibodies and fragments. DNA is cloned into a bacterial expressionsystem. One example of such a technique suitable for the practice ofthis invention uses a bacteriophage lambda vector system having a leadersequence that causes the expressed Fab protein to migrate to theperiplasmic space (between the bacterial cell membrane and the cellwall) or to be secreted. One can rapidly generate and screen greatnumbers of functional Fab fragments for those which bind IL-1β. SuchIL-1β binding agents (Fab fragments with specificity for an IL-1βpolypeptide) are specifically encompassed within the IL-1β bindingantibodies and fragments of the present invention.

The present IL-1 (e.g., IL-1β) binding antibodies and fragments can behumanized or human engineered antibodies. As used herein, a humanizedantibody, or antigen binding fragment thereof, is a recombinantpolypeptide that comprises a portion of an antigen binding site from anon-human antibody and a portion of the framework and/or constantregions of a human antibody. A human engineered antibody or antibodyfragment is a non-human (e.g., mouse) antibody that has been engineeredby modifying (e.g., deleting, inserting, or substituting) amino acids atspecific positions so as to reduce or eliminate any detectableimmunogenicity of the modified antibody in a human.

Humanized antibodies include chimeric antibodies and CDR-graftedantibodies. Chimeric antibodies are antibodies that include a non-humanantibody variable region linked to a human constant region. Thus, inchimeric antibodies, the variable region is mostly non-human, and theconstant region is human. Chimeric antibodies and methods for makingthem are described in Morrison, et al., Proc. Natl. Acad. Sci. USA, 81:6841-6855 (1984), Boulianne, et al., Nature, 312: 643-646 (1984), andPCT Application Publication WO 86/01533. Although, they can be lessimmunogenic than a mouse monoclonal antibody, administrations ofchimeric antibodies have been associated with human anti-mouse antibodyresponses (HAMA) to the non-human portion of the antibodies. Chimericantibodies can also be produced by splicing the genes from a mouseantibody molecule of appropriate antigen-binding specificity togetherwith genes from a human antibody molecule of appropriate biologicalactivity, such as the ability to activate human complement and mediateADCC. Morrison et al. (1984), Proc. Natl. Acad. Sci., 81: 6851;Neuberger et al. (1984), Nature, 312: 604. One example is thereplacement of a Fc region with that of a different isotype.

CDR-grafted antibodies are antibodies that include the CDRs from anon-human “donor” antibody linked to the framework region from a human“recipient” antibody. Generally, CDR-grafted antibodies include morehuman antibody sequences than chimeric antibodies because they includeboth constant region sequences and variable region (framework) sequencesfrom human antibodies. Thus, for example, a CDR-grafted humanizedantibody of the invention can comprise a heavy chain that comprises acontiguous amino acid sequence (e.g., about 5 or more, 10 or more, oreven 15 or more contiguous amino acid residues) from the frameworkregion of a human antibody (e.g., FR-1, FR-2, or FR-3 of a humanantibody) or, optionally, most or all of the entire framework region ofa human antibody. CDR-grafted antibodies and methods for making them aredescribed in, Jones et al., Nature, 321: 522-525 (1986), Riechmann etal., Nature, 332: 323-327 (1988), and Verhoeyen et al., Science, 239:1534-1536 (1988)). Methods that can be used to produce humanizedantibodies also are described in U.S. Pat. Nos. 4,816,567, 5,721,367,5,837,243, and 6,180,377. CDR-grafted antibodies are considered lesslikely than chimeric antibodies to induce an immune reaction againstnon-human antibody portions. However, it has been reported thatframework sequences from the donor antibodies are required for thebinding affinity and/or specificity of the donor antibody, presumablybecause these framework sequences affect the folding of theantigen-binding portion of the donor antibody. Therefore, when donor,non-human CDR sequences are grafted onto unaltered human frameworksequences, the resulting CDR-grafted antibody can exhibit, in somecases, loss of binding avidity relative to the original non-human donorantibody. See, e.g., Riechmann et al., Nature, 332: 323-327 (1988), andVerhoeyen et al., Science, 239: 1534-1536 (1988).

Human engineered antibodies include for example “veneered” antibodiesand antibodies prepared using HUMAN ENGINEERING™ technology (see forexample, U.S. Pat. Nos. 5,766,886 and 5,869,619). HUMAN ENGINEERING™technology is commercially available, and involves altering an non-humanantibody or antibody fragment, such as a mouse or chimeric antibody orantibody fragment, by making specific changes to the amino acid sequenceof the antibody so as to produce a modified antibody with reducedimmunogenicity in a human that nonetheless retains the desirable bindingproperties of the original non-human antibodies. Generally, thetechnique involves classifying amino acid residues of a non-human (e.g.,mouse) antibody as “low risk”, “moderate risk”, or “high risk” residues.The classification is performed using a global risk/reward calculationthat evaluates the predicted benefits of making particular substitution(e.g., for immunogenicity in humans) against the risk that thesubstitution will affect the resulting antibody's folding and/orantigen-binding properties. Thus, a low risk position is one for which asubstitution is predicted to be beneficial because it is predicted toreduce immunogenicity without significantly affecting antigen bindingproperties. A moderate risk position is one for which a substitution ispredicted to reduce immunogenicity, but is more likely to affect proteinfolding and/or antigen binding. High risk positions contain residuesmost likely to be involved in proper folding or antigen binding.Generally, low risk positions in a non-human antibody are substitutedwith human residues, high risk positions are rarely substituted, andhumanizing substitutions at moderate risk positions are sometimes made,although not indiscriminately. Positions with prolines in the non-humanantibody variable region sequence are usually classified as at leastmoderate risk positions.

The particular human amino acid residue to be substituted at a given lowor moderate risk position of a non-human (e.g., mouse) antibody sequencecan be selected by aligning an amino acid sequence from the non-humanantibody's variable regions with the corresponding region of a specificor consensus human antibody sequence. The amino acid residues at low ormoderate risk positions in the non-human sequence can be substituted forthe corresponding residues in the human antibody sequence according tothe alignment. Techniques for making human engineered proteins aredescribed in greater detail in Studnicka et al., Protein Engineering, 7:805-814 (1994), U.S. Pat. Nos. 5,766,886, 5,770,196, 5,821,123, and5,869,619, and PCT Application Publication WO 93/11794.

“Veneered” antibodies are non-human or humanized (e.g., chimeric orCDR-grafted antibodies) antibodies that have been engineered to replacecertain solvent-exposed amino acid residues so as to further reducetheir immunogenicity or enhance their function. As surface residues of achimeric antibody are presumed to be less likely to affect properantibody folding and more likely to elicit an immune reaction, veneeringof a chimeric antibody can include, for instance, identifyingsolvent-exposed residues in the non-human framework region of a chimericantibody and replacing at least one of them with the correspondingsurface residues from a human framework region. Veneering can beaccomplished by any suitable engineering technique, including the use ofthe above-described HUMAN ENGINEERING™ technology.

In a different approach, a recovery of binding avidity can be achievedby “de-humanizing” a CDR-grafted antibody. De-humanizing can includerestoring residues from the donor antibody's framework regions to theCDR grafted antibody, thereby restoring proper folding. Similar“de-humanization” can be achieved by (i) including portions of the“donor” framework region in the “recipient” antibody or (ii) graftingportions of the “donor” antibody framework region into the recipientantibody (along with the grafted donor CDRs).

For a further discussion of antibodies, humanized antibodies, humanengineered, and methods for their preparation, see Kontermann and Dubel,eds., Antibody Engineering, Springer, New York, N.Y., 2001.

Exemplary humanized or human engineered antibodies include IgG, IgM,IgE, IgA, and IgD antibodies. The present antibodies can be of any class(IgG, IgA, IgM, IgE, IgD, etc.) or isotype and can comprise a kappa orlambda light chain. For example, a human antibody can comprise an IgGheavy chain or defined fragment, such as at least one of isotypes, IgG1,IgG2, IgG3 or IgG4. As a further example, the present antibodies orfragments can comprise an IgG1 heavy chain and an IgG1 light chain.

The present antibodies and fragments can be human antibodies, such asantibodies which bind IL-1β polypeptides and are encoded by nucleic acidsequences which are naturally occurring somatic variants of humangermline immunoglobulin nucleic acid sequence, and fragments, syntheticvariants, derivatives and fusions thereof. Such antibodies may beproduced by any method known in the art, such as through the use oftransgenic mammals (such as transgenic mice) in which the nativeimmunoglobulin repertoire has been replaced with human V-genes in themammal chromosome. Such mammals appear to carry out VDJ recombinationand somatic hypermutation of the human germline antibody genes in anormal fashion, thus producing high affinity antibodies with completelyhuman sequences.

Human antibodies to target protein can also be produced using transgenicanimals that have no endogenous immunoglobulin production and areengineered to contain human immunoglobulin loci. For example, WO98/24893 discloses transgenic animals having a human Ig locus whereinthe animals do not produce functional endogenous immunoglobulins due tothe inactivation of endogenous heavy and light chain loci. WO 91/00906also discloses transgenic non-primate mammalian hosts capable ofmounting an immune response to an immunogen, wherein the antibodies haveprimate constant and/or variable regions, and wherein the endogenousimmunoglobulin encoding loci are substituted or inactivated. WO 96/30498and U.S. Pat. No. 6,091,001 disclose the use of the Cre/Lox system tomodify the immunoglobulin locus in a mammal, such as to replace all or aportion of the constant or variable region to form a modified antibodymolecule. WO 94/02602 discloses non-human mammalian hosts havinginactivated endogenous Ig loci and functional human Ig loci. U.S. Pat.No. 5,939,598 discloses methods of making transgenic mice in which themice lack endogenous heavy chains, and express an exogenousimmunoglobulin locus comprising one or more xenogeneic constant regions.See also, U.S. Pat. Nos. 6,114,598 6,657,103 and 6,833,268.

Using a transgenic animal described above, an immune response can beproduced to a selected antigenic molecule, and antibody producing cellscan be removed from the animal and used to produce hybridomas thatsecrete human monoclonal antibodies. Immunization protocols, adjuvants,and the like are known in the art, and are used in immunization of, forexample, a transgenic mouse as described in WO 96/33735. Thispublication discloses monoclonal antibodies against a variety ofantigenic molecules including IL-6, IL-8, TNFa, human CD4, L selectin,gp39, and tetanus toxin. The monoclonal antibodies can be tested for theability to inhibit or neutralize the biological activity orphysiological effect of the corresponding protein. WO 96/33735 disclosesthat monoclonal antibodies against IL-8, derived from immune cells oftransgenic mice immunized with IL-8, blocked IL-8 induced functions ofneutrophils. Human monoclonal antibodies with specificity for theantigen used to immunize transgenic animals are also disclosed in WO96/34096 and U.S. patent application no. 20030194404; and U.S. patentapplication no. 20030031667.

Additional transgenic animals useful to make monoclonal antibodiesinclude the Medarex HuMAb-MOUSE®, described in U.S. Pat. No. 5,770,429and Fishwild, et al. (Nat. Biotechnol. 14:845-851, 1996), which containsgene sequences from unrearranged human antibody genes that code for theheavy and light chains of human antibodies. Immunization of aHuMAb-MOUSE® enables the production of fully human monoclonal antibodiesto the target protein.

Also, Ishida et al. (Cloning Stem Cells. 4:91-102, 2002) describes theTransChromo Mouse (TCMOUSE™) which comprises megabase-sized segments ofhuman DNA and which incorporates the entire human immunoglobulin (hIg)loci. The TCMOUSE™ has a fully diverse repertoire of hlgs, including allthe subclasses of IgGs (IgG1-G4). Immunization of the TC MOUSE™ withvarious human antigens produces antibody responses comprising humanantibodies.

See also Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Yearin Immunol., 7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369,5,545,807; and U.S Patent Publication No. 20020199213. U.S. PatentPublication No. 20030092125 describes methods for biasing the immuneresponse of an animal to the desired epitope. Human antibodies may alsobe generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610and 5,229,275).

Human antibodies can also be generated through the in vitro screening ofantibody display libraries. See Hoogenboom et al. (1991), J. Mol. Biol.227: 381; and Marks et al. (1991), J. Mol. Biol. 222: 581. Variousantibody-containing phage display libraries have been described and maybe readily prepared. Libraries may contain a diversity of human antibodysequences, such as human Fab, Fv, and scFv fragments, that may bescreened against an appropriate target. Phage display libraries maycomprise peptides or proteins other than antibodies which may bescreened to identify selective binding agents of IL-1β.

The development of technologies for making repertoires of recombinanthuman antibody genes, and the display of the encoded antibody fragmentson the surface of filamentous bacteriophage, has provided a means formaking human antibodies directly. The antibodies produced by phagetechnology are produced as antigen binding fragments-usually Fv or Fabfragments-in bacteria and thus lack effector functions. Effectorfunctions can be introduced by one of two strategies: The fragments canbe engineered either into complete antibodies for expression inmammalian cells, or into bispecific antibody fragments with a secondbinding site capable of triggering an effector function.

The invention contemplates a method for producing target-specificantibody or antigen-binding portion thereof comprising the steps ofsynthesizing a library of human antibodies on phage, screening thelibrary with target protein or a portion thereof, isolating phage thatbind target, and obtaining the antibody from the phage. By way ofexample, one method for preparing the library of antibodies for use inphage display techniques comprises the steps of immunizing a non-humananimal comprising human immunoglobulin loci with target antigen or anantigenic portion thereof to create an immune response, extractingantibody producing cells from the immunized animal; isolating RNA fromthe extracted cells, reverse transcribing the RNA to produce cDNA,amplifying the cDNA using a primer, and inserting the cDNA into a phagedisplay vector such that antibodies are expressed on the phage.Recombinant target-specific antibodies of the invention may be obtainedin this way.

Phage-display processes mimic immune selection through the display ofantibody repertoires on the surface of filamentous bacteriophage, andsubsequent selection of phage by their binding to an antigen of choice.One such technique is described in WO 99/10494, which describes theisolation of high affinity and functional agonistic antibodies for MPLand msk receptors using such an approach. Antibodies of the inventioncan be isolated by screening of a recombinant combinatorial antibodylibrary, preferably a scFv phage display library, prepared using humanV_(L) and V_(H) cDNAs prepared from mRNA derived from human lymphocytes.Methodologies for preparing and screening such libraries are known inthe art. See e.g., U.S. Pat. No. 5,969,108. There are commerciallyavailable kits for generating phage display libraries (e.g., thePharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; andthe Stratagene SurfZAP™ phage display kit, catalog no. 240612). Thereare also other methods and reagents that can be used in generating andscreening antibody display libraries (see, e.g., Ladner et al. U.S. Pat.No. 5,223,409; Kang et al. PCT Publication No. WO 92/18619; Dower et al.PCT Publication No. WO 91/17271; Winter et al. PCT Publication No. WO92/20791; Markland et al. PCT Publication No. WO 92/15679; Breitling etal. PCT Publication No. WO 93/01288; McCafferty et al. PCT PublicationNo. WO 92/01047; Garrard et al. PCT Publication No. WO 92/09690; Fuchset al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;McCafferty et al., Nature (1990) 348:552-554; Griffiths et al. (1993)EMBO J 12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896;Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) Proc.Natl. Acad. Sci. USA 89:3576-3580; Garrad et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; andBarbas et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982.

In one embodiment, to isolate human antibodies specific for the targetantigen with the desired characteristics, a human V_(H) and V_(L)library are screened to select for antibody fragments having the desiredspecificity. The antibody libraries used in this method are preferablyscFv libraries prepared and screened as described herein and in the art(McCafferty et al., PCT Publication No. WO 92/01047, McCafferty et al.,(Nature 348:552-554, 1990); and Griffiths et al., (EMBO J 12:725-734,1993). The scFv antibody libraries preferably are screened using targetprotein as the antigen.

Alternatively, the Fd fragment (V_(H)-C_(H)1) and light chain(V_(L)-C_(L)) of antibodies are separately cloned by PCR and recombinedrandomly in combinatorial phage display libraries, which can then beselected for binding to a particular antigen. The Fab fragments areexpressed on the phage surface, i.e., physically linked to the genesthat encode them. Thus, selection of Fab by antigen binding co-selectsfor the Fab encoding sequences, which can be amplified subsequently.Through several rounds of antigen binding and re-amplification, aprocedure termed panning, Fab specific for the antigen are enriched andfinally isolated.

In 1994, an approach for the humanization of antibodies, called “guidedselection”, was described. Guided selection utilizes the power of thephage display technique for the humanization of mouse monoclonalantibody (See Jespers, L. S., et al., Bio/Technology 12, 899-903(1994)). For this, the Fd fragment of the mouse monoclonal antibody canbe displayed in combination with a human light chain library, and theresulting hybrid Fab library may then be selected with antigen. Themouse Fd fragment thereby provides a template to guide the selection.Subsequently, the selected human light chains are combined with a humanFd fragment library. Selection of the resulting library yields entirelyhuman Fab.

A variety of procedures have been described for deriving humanantibodies from phage-display libraries (See, for example, Hoogenboom etal., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol,222:581-597 (1991); U.S. Pat. Nos. 5,565,332 and 5,573,905; Clackson,T., and Wells, J. A., TIBTECH 12, 173-184 (1994)). In particular, invitro selection and evolution of antibodies derived from phage displaylibraries has become a powerful tool (See Burton, D. R., and Barbas III,C. F., Adv. Immunol. 57, 191-280 (1994); Winter, G., et al., Annu. Rev.Immunol. 12, 433-455 (1994); U.S. patent publication no. 20020004215 andWO 92/01047; U.S. patent publication no. 20030190317; and U.S. Pat. Nos.6,054,287 and 5,877,293.

Watkins, “Screening of Phage-Expressed Antibody Libraries by CaptureLift,” Methods in Molecular Biology, Antibody Phage Display: Methods andProtocols 178: 187-193 (2002), and U.S. patent publication no.20030044772, published Mar. 6, 2003, describe methods for screeningphage-expressed antibody libraries or other binding molecules by capturelift, a method involving immobilization of the candidate bindingmolecules on a solid support.

Fv fragments are displayed on the surface of phage, by the associationof one chain expressed as a phage protein fusion (e.g., with M13 geneIII) with the complementary chain expressed as a soluble fragment. It iscontemplated that the phage may be a filamentous phage such as one ofthe class I phages: fd, M13, f1, If1, Ike, ZJ/Z, Ff and one of the classII phages Xf, Pf1 and Pf3. The phage may be M13, or fd or a derivativethereof.

Once initial human V_(L) and V_(H) segments are selected, “mix andmatch” experiments, in which different pairs of the initially selectedV_(L) and V_(H) segments are screened for target binding, are performedto select preferred V_(L)/V_(H) pair combinations. Additionally, tofurther improve the quality of the antibody, the V_(L) and V_(H)segments of the preferred V_(L)/V_(H) pair(s) can be randomly mutated,preferably within the any of the CDR1, CDR2 or CDR3 region of V_(H)and/or V_(L), in a process analogous to the in vivo somatic mutationprocess responsible for affinity maturation of antibodies during anatural immune response. This in vitro affinity maturation can beaccomplished by amplifying V_(L) and V_(H) regions using PCR primerscomplimentary to the V_(H) CDR1, CDR2, and CDR3, or V_(L) CDR1, CDR2,and CDR3, respectively, which primers have been “spiked” with a randommixture of the four nucleotide bases at certain positions such that theresultant PCR products encode V_(L) and V_(H) segments into which randommutations have been introduced into the V_(H) and/or V_(L) CDR3 regions.These randomly mutated V_(L) and V_(H) segments can be rescreened forbinding to target antigen.

Following screening and isolation of an target specific antibody from arecombinant immunoglobulin display library, nucleic acid encoding theselected antibody can be recovered from the display package (e.g., fromthe phage genome) and subcloned into other expression vectors bystandard recombinant DNA techniques. If desired, the nucleic acid can befurther manipulated to create other antibody forms of the invention, asdescribed below. To express a recombinant human antibody isolated byscreening of a combinatorial library, the DNA encoding the antibody iscloned into a recombinant expression vector and introduced into amammalian host cell, as described herein.

It is contemplated that the phage display method may be carried out in amutator strain of bacteria or host cell. A mutator strain is a host cellwhich has a genetic defect which causes DNA replicated within it to bemutated with respect to its parent DNA. Example mutator strains areNR9046mutD5 and NR9046 mut Ti.

It is also contemplated that the phage display method may be carried outusing a helper phage. This is a phage which is used to infect cellscontaining a defective phage genome and which functions to complementthe defect. The defective phage genome can be a phagemid or a phage withsome function encoding gene sequences removed. Examples of helper phagesare M13K07, M13K07 gene III no. 3; and phage displaying or encoding abinding molecule fused to a capsid protein.

Antibodies are also generated via phage display screening methods usingthe hierarchical dual combinatorial approach as disclosed in WO 92/01047in which an individual colony containing either an H or L chain clone isused to infect a complete library of clones encoding the other chain (Lor H) and the resulting two-chain specific binding member is selected inaccordance with phage display techniques such as those describedtherein. This technique is also disclosed in Marks et al,(Bio/Technology, 10:779-783, 1992).

Methods for display of peptides on the surface of yeast and microbialcells have also been used to identify antigen specific antibodies. See,for example, U.S. Pat. No. 6,699,658. Antibody libraries may be attachedto yeast proteins, such as agglutinin, effectively mimicking the cellsurface display of antibodies by B cells in the immune system.

In addition to phage display methods, antibodies may be isolated usingribosome mRNA display methods and microbial cell display methods.Selection of polypeptide using ribosome display is described in Hanes etal., (Proc. Natl Acad Sci USA, 94:4937-4942, 1997) and U.S. Pat. Nos.5,643,768 and 5,658,754 issued to Kawasaki. Ribosome display is alsouseful for rapid large scale mutational analysis of antibodies. Theselective mutagenesis approach also provides a method of producingantibodies with improved activities that can be selected using ribosomaldisplay techniques.

The IL-1 (e.g., IL-1β) binding antibodies and fragments may comprise oneor more portions that do not bind IL-1β but instead are responsible forother functions, such as circulating half-life, direct cytotoxic effect,detectable labeling, or activation of the recipient's endogenouscomplement cascade or endogenous cellular cytotoxicity. The antibodiesor fragments may comprise all or a portion of the constant region andmay be of any isotype, including IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG(e.g. IgG1, IgG2, IgG3 or IgG4), or IgM. In addition to, or instead of,comprising a constant region, antigen-binding compounds of the inventionmay include an epitope tag, a salvage receptor epitope, a label moietyfor diagnostic or purification purposes, or a cytotoxic moiety such as aradionuclide or toxin.

The constant region (when present) of the present antibodies andfragments may be of the γ1, γ2, γ3, γ4, μ, β2, or δ or ε type,preferably of the γ type, more preferably of the y, type, whereas theconstant part of a human light chain may be of the κ or λ type (whichincludes the λ₁, λ₂ and λ₃ subtypes) but is preferably of the κ type.

Variants also include antibodies or fragments comprising a modified Fcregion, wherein the modified Fc region comprises at least one amino acidmodification relative to a wild-type Fc region. The variant Fc regionmay be designed, relative to a comparable molecule comprising thewild-type Fc region, so as to bind Fc receptors with a greater or lesseraffinity.

For example, the present IL-1β binding antibodies and fragments maycomprise a modified Fc region. Fc region refers to naturally-occurringor synthetic polypeptides homologous to the IgG C-terminal domain thatis produced upon papain digestion of IgG. IgG Fc has a molecular weightof approximately 50 kD. In the present antibodies and fragments, anentire Fc region can be used, or only a half-life enhancing portion. Inaddition, many modifications in amino acid sequence are acceptable, asnative activity is not in all cases necessary or desired.

The Fc region can be mutated, if desired, to inhibit its ability to fixcomplement and bind the Fc receptor with high affinity. For murine IgGFc, substitution of Ala residues for Glu 318, Lys 320, and Lys 322renders the protein unable to direct ADCC. Substitution of Glu for Leu235 inhibits the ability of the protein to bind the Fc receptor withhigh affinity. Various mutations for human IgG also are known (see,e.g., Morrison et al., 1994, The Immunologist 2: 119 124 and Brekke etal., 1994, The Immunologist 2: 125).

In some embodiments, the present an antibodies or fragments are providedwith a modified Fc region where a naturally-occurring Fc region ismodified to increase the half-life of the antibody or fragment in abiological environment, for example, the serum half-life or a half-lifemeasured by an in vitro assay. Methods for altering the original form ofa Fc region of an IgG also are described in U.S. Pat. No. 6,998,253.

In certain embodiments, it may be desirable to modify the antibody orfragment in order to increase its serum half-life, for example, addingmolecules such as PEG or other water soluble polymers, includingpolysaccharide polymers, to antibody fragments to increase thehalf-life. This may also be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment (e.g., bymutation of the appropriate region in the antibody fragment or byincorporating the epitope into a peptide tag that is then fused to theantibody fragment at either end or in the middle, e.g., by DNA orpeptide synthesis) (see, International Publication No. WO96/32478).Salvage receptor binding epitope refers to an epitope of the Fc regionof an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, or IgG₄) that is responsiblefor increasing the in vivo serum half-life of the IgG molecule.

A salvage receptor binding epitope can include a region wherein any oneor more amino acid residues from one or two loops of a Fc domain aretransferred to an analogous position of the antibody fragment. Even morepreferably, three or more residues from one or two loops of the Fcdomain are transferred. Still more preferred, the epitope is taken fromthe CH2 domain of the Fc region (e.g., of an IgG) and transferred to theCH1, CH3, or V_(H) region, or more than one such region, of theantibody. Alternatively, the epitope is taken from the CH2 domain of theFc region and transferred to the C_(L) region or V_(L) region, or both,of the antibody fragment. See also International applications WO97/34631 and WO 96/32478 which describe Fc variants and theirinteraction with the salvage receptor.

Mutation of residues within Fc receptor binding sites can result inaltered effector function, such as altered ADCC or CDC activity, oraltered half-life. Potential mutations include insertion, deletion orsubstitution of one or more residues, including substitution withalanine, a conservative substitution, a non-conservative substitution,or replacement with a corresponding amino acid residue at the sameposition from a different IgG subclass (e.g. replacing an IgG1 residuewith a corresponding IgG2 residue at that position). For example it hasbeen reported that mutating the serine at amino acid position 241 inIgG4 to proline (found at that position in IgG1 and IgG2) led to theproduction of a homogeneous antibody, as well as extending serumhalf-life and improving tissue distribution compared to the originalchimeric IgG4. (Angal et al., Mol Immunol. 30:105-8, 1993).

Antibody fragments are portions of an intact full length antibody, suchas an antigen binding or variable region of the intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; linear antibodies; single-chain antibody molecules(e.g., scFv); multispecific antibody fragments such as bispecific,trispecific, and multispecific antibodies (e.g., diabodies, triabodies,tetrabodies); minibodies; chelating recombinant antibodies; tribodies orbibodies; intrabodies; nanobodies; small modular immunopharmaceuticals(SMIP), adnectins, binding-domain immunoglobulin fusion proteins;camelized antibodies; V_(HH) containing antibodies; and any otherpolypeptides formed from antibody fragments.

The present invention includes IL-1β binding antibody fragmentscomprising any of the foregoing heavy or light chain sequences and whichbind IL-1β. The term fragments as used herein refers to any 3 or morecontiguous amino acids (e.g., 4 or more, 5 or more 6 or more, 8 or more,or even 10 or more contiguous amino acids) of the antibody andencompasses Fab, Fab′, F(ab′)₂, and F(v) fragments, or the individuallight or heavy chain variable regions or portion thereof. IL-1β bindingfragments include, for example, Fab, Fab′, F(ab′)₂, Fv and scFv. Thesefragments lack the Fc fragment of an intact antibody, clear more rapidlyfrom the circulation, and can have less non-specific tissue binding thanan intact antibody. See Wahl et al. (1983), J. Nucl. Med., 24: 316-25.These fragments can be produced from intact antibodies using well knownmethods, for example by proteolytic cleavage with enzymes such as papain(to produce Fab fragments) or pepsin (to produce F(ab′)₂ fragments).

In vitro and cell based assays are well described in the art for use indetermining binding of IL-1β to IL-1 receptor type I (IL-1R1), includingassays that determining in the presence of molecules (such asantibodies, antagonists, or other inhibitors) that bind to IL-1β orIL-1RI. (see for example Evans et al., (1995), J. Biol. Chem.270:11477-11483; Vigers et al., (2000), J. Biol. Chem. 275:36927-36933;Yanofsky et al., (1996), Proc. Natl. Acad. Sci. USA 93:7381-7386;Fredericks et al., (2004), Protein Eng. Des. Sel. 17:95-106; Slack etal., (1993), J. Biol. Chem. 268:2513-2524; Smith et al., (2003),Immunity 18:87-96; Vigers et al., (1997), Nature 386:190-194; Ruggieroet al., (1997), J. Immunol. 158:3881-3887; Guo et al., (1995), J. Biol.Chem. 270:27562-27568; Svenson et al., (1995), Eur. J. Immunol.25:2842-2850; Arend et al., (1994), J. Immunol. 153:4766-4774).Recombinant IL-1 receptor type I, including human IL-1 receptor type I,for such assays is readily available from a variety of commercialsources (see for example R&D Systems, SIGMA). IL-1 receptor type I alsocan be expressed from an expression construct or vector introduced intoan appropriate host cell using standard molecular biology andtransfection techniques known in the art. The expressed IL-1 receptortype I may then be isolated and purified for use in binding assays, oralternatively used directly in a cell associated form.

For example, the binding of IL-1β to IL-1 receptor type I may bedetermined by immobilizing an IL-1β binding antibody, contacting IL-1βwith the immobilized antibody and determining whether the IL-1β wasbound to the antibody, and contacting a soluble form of IL-1RI with thebound IL-1β/antibody complex and determining whether the soluble IL-1RIwas bound to the complex. The protocol may also include contacting thesoluble IL-1RI with the immobilized antibody before the contact withIL-1β, to confirm that the soluble IL-1RI does not bind to theimmobilized antibody. This protocol can be performed using a Biacore®instrument for kinetic analysis of binding interactions. Such a protocolcan also be employed to determine whether an antibody or other moleculepermits or blocks the binding of IL-1β to IL-1 receptor type I.

For other IL-1β/IL-1RI binding assays, the permitting or blocking ofIL-1β binding to IL-1 receptor type I may be determined by comparing thebinding of IL-1β to IL-1RI in the presence or absence of IL-1βantibodies or IL-1β binding fragments thereof. Blocking is identified inthe assay readout as a designated reduction of IL-1β binding to IL-1receptor type I in the presence of anti-IL-1β antibodies or IL-1βbinding fragments thereof, as compared to a control sample that containsthe corresponding buffer or diluent but not an IL-1β antibody or IL-1βbinding fragment thereof. The assay readout may be qualitatively viewedas indicating the presence or absence of blocking, or may bequantitatively viewed as indicating a percent or fold reduction inbinding due to the presence of the antibody or fragment.

Alternatively or additionally, when an IL-1β binding antibody or IL-1βbinding fragment substantially blocks IL-1β binding to IL-1RI, the IL-1βbinding to IL-1RI is reduced by at least 10-fold, alternatively at leastabout 20-fold, alternatively at least about 50-fold, alternatively atleast about 100-fold, alternatively at least about 1000-fold,alternatively at least about 10000-fold, or more, compared to binding ofthe same concentrations of IL-1β and IL-1RI in the absence of theantibody or fragment. As another example, when an IL-1β binding antibodyor IL-1β binding fragment substantially permits IL-1β binding to IL-1RI,the IL-1β binding to IL-1RI is at least about 90%, alternatively atleast about 95%, alternatively at least about 99%, alternatively atleast about 99.9%, alternatively at least about 99.99%, alternatively atleast about 99.999%, alternatively at least about 99.9999%,alternatively substantially identical to binding of the sameconcentrations of IL-1β and IL-1RI in the absence of the antibody orfragment.

The present invention may in certain embodiments encompass IL-1β bindingantibodies or IL-1β binding fragments that bind to the same epitope orsubstantially the same epitope as one or more of the exemplaryantibodies described herein. Alternatively or additionally, the IL-1βbinding antibodies or IL-1β binding fragments compete with the bindingof an antibody having variable region sequences of AB7, described inU.S. application Ser. No. 11/472,813 or WO 2007/002261 (sequences shownbelow). As an example, when an IL-1β binding antibody or IL-1β bindingfragment competes with the binding of an antibody having the light chainvariable region of SEQ ID NO:5 and the heavy chain variable region ofSEQ ID NO:6, binding of an antibody having the light chain variableregion of SEQ ID NO:5 and the heavy chain variable region of SEQ ID NO:6to IL-1β may be reduced by at least about 2-fold, alternatively at leastabout 5-fold, alternatively at least about 10-fold, alternatively atleast about 20-fold, alternatively at least about 50-fold, alternativelyat least about 100-fold, alternatively at least about 1000-fold,alternatively at least about 10000-fold, or more, if the binding ismeasured in the presence of the IL-1β binding antibody or IL-1β bindingfragment. The IL-1β binding antibody or IL-1β binding fragment may bepresent in excess of the antibody having the light chain variable regionof SEQ ID NO:5 and the heavy chain variable region of SEQ ID NO:6, forexample an excess of least about 2-fold, alternatively at least about5-fold, alternatively at least about 10-fold, alternatively at leastabout 20-fold, alternatively at least about 50-fold, alternatively atleast about 100-fold, alternatively at least about 1000-fold,alternatively at least about 10000-fold. Alternatively or additionally,the present invention encompasses IL-1β binding antibodies and fragmentsthat bind to an epitope contained in the amino acid sequenceESVDPKNYPKKKMEKRFVFNKIE (SEQ ID NO: 1), an epitope that the antibodiesdesignated AB5 and AB7 (U.S. application Ser. No. 11/472,813, WO2007/002261) bind to. As contemplated herein, one can readily determineif an IL-1β binding antibody or fragment binds to the same epitope orsubstantially the same epitope as one or more of the exemplaryantibodies, such as for example the antibody designated AB7, using anyof several known methods in the art.

For example, the key amino acid residues (epitope) bound by an IL-1βbinding antibody or fragment may be determined using a peptide array,such as for example, a PepSpot™ peptide array (JPT Peptide Technologies,Berlin, Germany), wherein a scan of twelve amino-acid peptides, spanningthe entire IL-1β amino acid sequence, each peptide overlapping by 11amino acid to the previous one, is synthesized directly on a membrane.The membrane carrying the peptides is then probed with the antibody forwhich epitope binding information is sought, for example at aconcentration of 2 pg/ml, for 2 hr at room temperature. Binding ofantibody to membrane bound peptides may be detected using a secondaryHRP-conjugated goat anti-human (or mouse, when appropriate) antibody,followed by enhanced chemiluminescence (ECL). The peptides spot(s)corresponding to particular amino acid residues or sequences of themature IL-1β protein, and which score positive for antibody binding, areindicative of the epitope bound by the particular antibody.

Alternatively or in addition, antibody competition experiments may beperformed and such assays are well known in the art. For example, todetermine if an antibody or fragment binds to an epitope contained in apeptide sequence comprising the amino acids ESVDPKNYPKKKMEKRFVFNKIE (SEQID NO: 1), which corresponds to residues 83-105 of the mature IL-1βprotein, an antibody of unknown specificity may be compared with any ofthe exemplary of antibodies (e.g., AB7) of the present invention thatare known to bind an epitope contained within this sequence. Bindingcompetition assays may be performed, for example, using a Biacore®instrument for kinetic analysis of binding interactions or by ELISA. Insuch an assay, the antibody of unknown epitope specificity is evaluatedfor its ability to compete for binding against the known comparatorantibody (e.g., AB7). Competition for binding to a particular epitope isdetermined by a reduction in binding to the IL-1β epitope of at leastabout 50%, or at least about 70%, or at least about 80%, or at leastabout 90%, or at least about 95%, or at least about 99% or about 100%for the known comparator antibody (e.g., AB7) and is indicative ofbinding to substantially the same epitope.

In view of the identification in this disclosure of IL-1β bindingregions in exemplary antibodies and/or epitopes recognized by thedisclosed antibodies, it is contemplated that additional antibodies withsimilar binding characteristics and therapeutic or diagnostic utilitycan be generated that parallel the embodiments of this disclosure.

Antigen-binding fragments of an antibody include fragments that retainthe ability to specifically bind to an antigen, generally by retainingthe antigen-binding portion of the antibody. It is well established thatthe antigen-binding function of an antibody can be performed byfragments of a full-length antibody. Examples of antigen-bindingportions include (i) a Fab fragment, which is a monovalent fragmentconsisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)² fragment,which is a bivalent fragment comprising two Fab fragments linked by adisulfide bridge at the hinge region; (iii) a Fd fragment which is theVH and CH1 domains; (iv) a Fv fragment which is the VL and VH domains ofa single arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which is a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Single chain antibodies arealso encompassed within the term antigen-binding portion of an antibody.The IL-1β binding antibodies and fragments of the present invention alsoencompass monovalent or multivalent, or monomeric or multimeric (e.g.tetrameric), CDR-derived binding domains with or without a scaffold (forexample, protein or carbohydrate scaffolding).

The present IL-1β binding antibodies or fragments may be part of alarger immunoadhesion molecules, formed by covalent or non-covalentassociation of the antibody or antibody portion with one or more otherproteins or peptides. Examples of such immunoadhesion molecules includeuse of the streptavidin core region to make a tetrameric scFv molecule(Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas6:93-101) and use of a cysteine residue, a marker peptide and aC-terminal polyhistidine tag to make bivalent and biotinylated scFvmolecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058).Antibodies and fragments comprising immunoadhesion molecules can beobtained using standard recombinant DNA techniques, as described herein.Preferred antigen binding portions are complete domains or pairs ofcomplete domains.

The IL-1β binding antibodies and fragments of the present invention alsoencompass domain antibody (dAb) fragments (Ward et al., Nature341:544-546, 1989) which consist of a V_(H) domain. The IL-1β bindingantibodies and fragments of the present invention also encompassdiabodies, which are bivalent antibodies in which V_(H) and V_(L)domains are expressed on a single polypeptide chain, but using a linkerthat is too short to allow for pairing between the two domains on thesame chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., EP 404,097; WO 93/11161; Holliger et al., Proc. Natl. Acad. Sci.USA 90:6444-6448, 1993, and Poljak et al., Structure 2:1121-1123, 1994).Diabodies can be bispecific or monospecific.

The IL-1β binding antibodies and fragments of the present invention alsoencompass single-chain antibody fragments (scFv) that bind to IL-1β. AnscFv comprises an antibody heavy chain variable region (V_(H)) operablylinked to an antibody light chain variable region (V_(L)) wherein theheavy chain variable region and the light chain variable region,together or individually, form a binding site that binds IL-1β. An scFvmay comprise a V_(H) region at the amino-terminal end and a V_(L) regionat the carboxy-terminal end. Alternatively, scFv may comprise a V_(L)region at the amino-terminal end and a V_(H) region at thecarboxy-terminal end. Furthermore, although the two domains of the Fvfragment, V_(L) and V_(H), are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(L) and V_(H)regions pair to form monovalent molecules (known as single chain Fv(scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston etal. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).

An scFv may optionally further comprise a polypeptide linker between theheavy chain variable region and the light chain variable region. Suchpolypeptide linkers generally comprise between 1 and 50 amino acids,alternatively between 3 and 12 amino acids, alternatively 2 amino acids.An example of a linker peptide for linking heavy and light chains in anscFv comprises the 5 amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:2). Other examples comprise one or more tandem repeats of this sequence(for example, a polypeptide comprising two to four repeats ofGly-Gly-Gly-Gly-Ser (SEQ ID NO: 2) to create linkers.

The IL-1β binding antibodies and fragments of the present invention alsoencompass heavy chain antibodies (HCAb). Exceptions to the H₂L₂structure of conventional antibodies occur in some isotypes of theimmunoglobulins found in camelids (camels, dromedaries and llamas;Hamers-Casterman et al., 1993 Nature 363: 446; Nguyen et al., 1998 J.Mol. Biol. 275: 413), wobbegong sharks (Nuttall et al., Mol Immunol.38:313-26, 2001), nurse sharks (Greenberg et al., Nature 374:168-73,1995; Roux et al., 1998 Proc. Nat. Acad. Sci. USA 95: 11804), and in thespotted ratfish (Nguyen, et al., “Heavy-chain antibodies in Camelidae; acase of evolutionary innovation,” 2002 Immunogenetics 54(1): 39-47).These antibodies can apparently form antigen-binding regions using onlyheavy chain variable regions, in that these functional antibodies aredimers of heavy chains only (referred to as “heavy-chain antibodies” or“HCAbs”). Accordingly, some embodiments of the present IL-1β bindingantibodies and fragments may be heavy chain antibodies that specificallybind to IL-1β. For example, heavy chain antibodies that are a class ofIgG and devoid of light chains are produced by animals of the genusCamelidae which includes camels, dromedaries and llamas(Hamers-Casterman et al., Nature 363:446-448 (1993)). HCAbs have amolecular weight of about 95 kDa instead of the about 160 kDa molecularweight of conventional IgG antibodies. Their binding domains consistonly of the heavy-chain variable domains, often referred to as V_(HH) todistinguish them from conventional V_(H). Muyldermans et al., J. Mol.Recognit. 12:131-140 (1999). The variable domain of the heavy-chainantibodies is sometimes referred to as a nanobody (Cortez-Retamozo etal., Cancer Research 64:2853-57, 2004). A nanobody library may begenerated from an immunized dromedary as described in Conrath et al.,(Antimicrob Agents Chemother 45: 2807-12, 2001) or using recombinantmethods.

Since the first constant domain (C_(H1)) is absent (spliced out duringmRNA processing due to loss of a splice consensus signal), the variabledomain (V_(HH)) is immediately followed by the hinge region, the C_(H2)and the C_(H3) domains (Nguyen et al., Mol. Immunol. 36:515-524 (1999);Woolven et al., Immunogenetics 50:98-101 (1999)). Camelid V_(HH)reportedly recombines with IgG2 and IgG3 constant regions that containhinge, CH2, and CH3 domains and lack a CH1 domain (Hamers-Casterman etal., supra). For example, llama IgG1 is a conventional (H₂L₂) antibodyisotype in which V_(H) recombines with a constant region that containshinge, CH1, CH2 and CH3 domains, whereas the llama IgG2 and IgG3 areheavy chain-only isotypes that lack CH1 domains and that contain nolight chains.

Although the HCAbs are devoid of light chains, they have anantigen-binding repertoire. The genetic generation mechanism of HCAbs isreviewed in Nguyen et al. Adv. Immunol 79:261-296 (2001) and Nguyen etal., Immunogenetics 54:39-47 (2002). Sharks, including the nurse shark,display similar antigen receptor-containing single monomeric V-domains.Irving et al., J. Immunol. Methods 248:31-45 (2001); Roux et al., Proc.Natl. Acad. Sci. USA 95:11804 (1998).

V_(HH)s comprise small intact antigen-binding fragments (for example,fragments that are about 15 kDa, 118-136 residues). Camelid V_(HH)domains have been found to bind to antigen with high affinity (Desmyteret al., J. Biol. Chem. 276:26285-90, 2001), with V_(HH) affinitiestypically in the nanomolar range and comparable with those of Fab andscFv fragments. V_(HH)s are highly soluble and more stable than thecorresponding derivatives of scFv and Fab fragments. V_(H) fragmentshave been relatively difficult to produce in soluble form, butimprovements in solubility and specific binding can be obtained whenframework residues are altered to be more V_(HH)-like. (See, forexample, Reichman et al., J Immunol Methods 1999, 231:25-38.) V_(HH)scarry amino acid substitutions that make them more hydrophilic andprevent prolonged interaction with BiP (immunoglobulin heavy-chainbinding protein), which normally binds to the H-chain in the EndoplasmicReticulum (ER) during folding and assembly, until it is displaced by theL-chain. Because of the V_(HH)s' increased hydrophilicity, secretionfrom the ER is improved.

Functional V_(HH)s may be obtained by proteolytic cleavage of HCAb of animmunized camelid, by direct cloning of V_(HH) genes from B-cells of animmunized camelid resulting in recombinant V_(HH)s, or from naive orsynthetic libraries. V_(HH)s with desired antigen specificity may alsobe obtained through phage display methodology. Using V_(HH)s in phagedisplay is much simpler and more efficient compared to Fabs or scFvs,since only one domain needs to be cloned and expressed to obtain afunctional antigen-binding fragment. Muyldermans, Biotechnol. 74:277-302(2001); Ghahroudi et al., FEBS Lett. 414:521-526 (1997); and van derLinden et al., J. Biotechnol. 80:261-270 (2000). Methods for generatingantibodies having camelid heavy chains are also described in U.S. PatentPublication Nos. 20050136049 and 20050037421.

Ribosome display methods may be used to identify and isolate scFv and/orV_(HH) molecules having the desired binding activity and affinity.Irving et al., J. Immunol. Methods 248:31-45 (2001). Ribosome displayand selection has the potential to generate and display large libraries(10¹⁴).

Other embodiments provide V_(HH)-like molecules generated through theprocess of camelisation, by modifying non-Camelidae V_(H)s, such ashuman V_(HH)S, to improve their solubility and prevent non-specificbinding. This is achieved by replacing residues on the V_(L)S side ofV_(H)s with V_(HH)-like residues, thereby mimicking the more solubleV_(HH) fragments. Camelised V_(H) fragments, particularly those based onthe human framework, are expected to exhibit a greatly reduced immuneresponse when administered in vivo to a patient and, accordingly, areexpected to have significant advantages for therapeutic applications.Davies et al., FEBS Lett. 339:285-290 (1994); Davies et al., ProteinEng. 9:531-537 (1996); Tanha et al., J. Biol. Chem. 276:24774-24780(2001); and Riechmann et al., Immunol. Methods 231:25-38 (1999).

A wide variety of expression systems are available for the production ofIL-1β fragments including Fab fragments, scFv, and V_(HH)S. For example,expression systems of both prokaryotic and eukaryotic origin may be usedfor the large-scale production of antibody fragments and antibody fusionproteins. Particularly advantageous are expression systems that permitthe secretion of large amounts of antibody fragments into the culturemedium.

Production of bispecific Fab-scFv (“bibody”) and trispecificFab-(scFv)(2) (“tribody”) are described in Schoonjans et al. (J Immunol.165:7050-57, 2000) and Willems et al. (J Chromatogr B Analyt TechnolBiomed Life Sci. 786:161-76, 2003). For bibodies or tribodies, a scFvmolecule is fused to one or both of the VL-CL (L) and VH-CH₁ (Fd)chains, e.g., to produce a tribody two scFvs are fused to C-term of Fabwhile in a bibody one scFv is fused to C-term of Fab. A “minibody”consisting of scFv fused to CH3 via a peptide linker (hingeless) or viaan IgG hinge has been described in Olafsen, et al., Protein Eng Des Sel.2004 April; 17(4):315-23.

Intrabodies are single chain antibodies which demonstrate intracellularexpression and can manipulate intracellular protein function (Biocca, etal., EMBO J. 9:101-108, 1990; Colby et al., Proc Natl Acad Sci USA.101:17616-21, 2004). Intrabodies, which comprise cell signal sequenceswhich retain the antibody construct in intracellular regions, may beproduced as described in Mhashilkar et al (EMBO J 14:1542-51, 1995) andWheeler et al. (FASEB J. 17:1733-5. 2003). Transbodies arecell-permeable antibodies in which a protein transduction domains (PTD)is fused with single chain variable fragment (scFv) antibodies Heng etal., (Med Hypotheses. 64:1105-8, 2005).

The IL-1β binding antibodies and fragments of the present invention alsoencompass antibodies that are SMIPs or binding domain immunoglobulinfusion proteins specific for target protein. These constructs aresingle-chain polypeptides comprising antigen binding domains fused toimmunoglobulin domains necessary to carry out antibody effectorfunctions. See e.g., WO03/041600, U.S. Patent publication 20030133939and US Patent Publication 20030118592.

The IL-1β binding antibodies and fragments of the present invention alsoencompass immunoadhesins. One or more CDRs may be incorporated into amolecule either covalently or noncovalently to make it an immunoadhesin.An immunoadhesin may incorporate the CDR(s) as part of a largerpolypeptide chain, may covalently link the CDR(s) to another polypeptidechain, or may incorporate the CDR(s) noncovalently. The CDRs disclosedherein permit the immunoadhesin to specifically bind to IL-1β.

The IL-1β binding antibodies and fragments of the present invention alsoencompass antibody mimics comprising one or more IL-1β binding portionsbuilt on an organic or molecular scaffold (such as a protein orcarbohydrate scaffold). Proteins having relatively definedthree-dimensional structures, commonly referred to as protein scaffolds,may be used as reagents for the design of antibody mimics. Thesescaffolds typically contain one or more regions which are amenable tospecific or random sequence variation, and such sequence randomizationis often carried out to produce libraries of proteins from which desiredproducts may be selected. For example, an antibody mimic can comprise achimeric non-immunoglobulin binding polypeptide having animmunoglobulin-like domain containing scaffold having two or moresolvent exposed loops containing a different CDR from a parent antibodyinserted into each of the loops and exhibiting selective bindingactivity toward a ligand bound by the parent antibody.Non-immunoglobulin protein scaffolds have been proposed for obtainingproteins with novel binding properties. (Tramontano et al., J. Mol.Recognit. 7:9, 1994; McConnell and Hoess, J. Mol. Biol. 250:460, 1995).Other proteins have been tested as frameworks and have been used todisplay randomized residues on alpha helical surfaces (Nord et al., Nat.Biotechnol. 15:772, 1997; Nord et al., Protein Eng. 8:601, 1995), loopsbetween alpha helices in alpha helix bundles (Ku and Schultz, Proc.Natl. Acad. Sci. USA 92:6552, 1995), and loops constrained by disulfidebridges, such as those of the small protease inhibitors (Markland etal., Biochemistry 35:8045, 1996; Markland et al., Biochemistry 35:8058,1996; Rottgen and Collins, Gene 164:243, 1995; Wang et al., J. Biol.Chem. 270:12250, 1995). Methods for employing scaffolds for antibodymimics are disclosed in U.S. Pat. No. 5,770,380 and US PatentPublications 2004/0171116, 2004/0266993, and 2005/0038229.

Preferred IL-1β antibodies or antibody fragments for use in accordancewith the invention generally bind to human IL-1β with high affinity(e.g., as determined with BIACORE), such as for example with anequilibrium binding dissociation constant (Ko) for IL-1β of about 10 nMor less, about 5 nM or less, about 1 nM or less, about 500 pM or less,or more preferably about 250 pM or less, about 100 pM or less, about 50pM or less, about 25 pM or less, about 10 pM or less, about 5 pM orless, about 3 pM or less about 1 pM or less, about 0.75 pM or less,about 0.5 pM or less, or about 0.3 pM or less. The dissociation constantmay be measured using Biacore (GE Healthcare), and measurement usingBiacore may be preferred when the dissociation constant is greater thanabout 10 pM. Alternatively or in addition, the dissociation constant maybe measured using KinExA (Sapidyne Instruments, Inc), and measurementusing KinExA may be preferred when the dissociation constant is lessthan about 10 pM.

Antibodies or fragments of the present invention may, for example, bindto IL-1β with an IC₅₀ of about 10 nM or less, about 5 nM or less, about2 nM or less, about 1 nM or less, about 0.75 nM or less, about 0.5 nM orless, about 0.4 nM or less, about 0.3 nM or less, or even about 0.2 nMor less, as determined by enzyme linked immunosorbent assay (ELISA).Preferably, the antibody or antibody fragment of the present inventiondoes not cross-react with any target other than IL-1. For example, thepresent antibodies and fragments may bind to IL-1β, but do notdetectably bind to IL-1α, or have at least about 100 times (e.g., atleast about 150 times, at least about 200 times, or even at least about250 times) greater selectivity in its binding of IL-1β relative to itsbinding of IL-1α. Antibodies or fragments used according to theinvention may, in certain embodiments, inhibit IL-1β induced expressionof serum IL-6 in an animal by at least 50% (e.g., at least 60%, at least70%, or even at least 80%) as compared to the level of serum IL-6 in anIL-1β stimulated animal that has not been administered an antibody orfragment of the invention. Antibodies may bind IL-1β but permit orsubstantially permit the binding of the bound IL-1β ligand to IL-1receptor type I (IL-1RI). In contrast to many known IL-1β bindingantibodies that block or substantially interfere with binding of IL-1βto IL-1RI, the antibodies designated AB5 and AB7 (U.S. application Ser.No. 11/472,813, WO 2007/002261) selectively bind to the IL-1β ligand,but permit the binding of the bound IL-1β ligand to IL-1RI. For example,the antibody designated AB7 binds to an IL-1β epitope but still permitsthe bound IL-1β to bind to IL-1RI. In certain embodiments, the antibodymay decrease the affinity of interaction of bound IL-1β to bind toIL-1RI. Accordingly, the invention provides, in a related aspect, use ofan IL-1β binding antibody or IL-1β binding antibody fragment that has atleast one of the aforementioned characteristics. Any of the foregoingantibodies, antibody fragments, or polypeptides of the invention can behumanized or human engineered, as described herein.

A variety of IL-1 (e.g., IL-1β) antibodies and fragments known in theart may be used according the methods provided herein, including forexample antibodies described in or derived using methods described inthe following patents and patent applications: U.S. Pat. No. 4,935,343;US 2003/0026806; US 2003/0124617 (e.g., antibody AAL160); WO 2006/081139(e.g., antibody 9.5.2); WO 03/034984; WO 95/01997 (e.g., antibodySK48-E26 VTKY); WO 02/16436 (e.g., antibody ACZ 885); WO 03/010282(e.g., antibody Hu007); WO 03/073982 (e.g., antibody N55S), WO2004/072116, WO 2004/067568, EP 0 267 611 B1, EP 0 364 778 B1, and U.S.application Ser. No. 11/472,813. As a non-limiting example, antibodiesAB5 and AB7 (U.S. application Ser. No. 11/472,813, WO2007/002261) may beused in accordance with the invention. Variable region sequences of AB5and AB7 (also referred to as XOMA 052) are as follows:

AB5 LIGHT CHAIN (SEQ ID NO: 3)DIQMTQTTSSLSASLGDRVTISCRASQDISNYLSWYQQKPDGTVKLLIYYTSKLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCLQGKMLPWTF GGGTKLEIK

The underlined sequences depict (from left to right) CDR1, 2 and 3.

HEAVY CHAIN (SEQ ID NO: 4)QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDGDESYNPSLKTQLTISKDTSRNQVFLKITSVDTVDTATYFCA RNRYDPPWFVDWGQGTLVTVSS

The underlined sequences depict (from left to right) CDR1, 2 and 3.

AB7 LIGHT CHAIN (SEQ ID NO: 5)DIQMTQSTSSLSASVGDRVTITCRASQDISNYLSWYQQKPGKAVKLLIYYTSKLHSGVPSRFSGSGSGTDYTLTISSLQQEDFATYFCLQGKMLPWTF GQGTKLEIK

The underlined sequences depict (from left to right) CDR1, 2 and 3.

HEAVY CHAIN (SEQ ID NO: 6)QVQLQESGPGLVKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDGDESYNPSLKSRLTISKDTSKNQVSLKITSVTAADTAVYFCA RNRYDPPWFVDWGQGTLVTVSS

The underlined sequences depict (from left to right) CDR1, 2 and 3.

The antibodies and antibody fragments described herein can be preparedby any suitable method. Suitable methods for preparing such antibodiesand antibody fragments are known in the art. Other methods for preparingthe antibodies and antibody fragments are as described herein as part ofthe invention. The antibody, antibody fragment, or polypeptide of theinvention, as described herein, can be isolated or purified to anydegree. As used herein, an isolated compound is a compound that has beenremoved from its natural environment. A purified compound is a compoundthat has been increased in purity, such that the compound exists in aform that is more pure than it exists (i) in its natural environment or(ii) when initially synthesized and/or amplified under laboratoryconditions, wherein “purity” is a relative term and does not necessarilymean “absolute purity.”

Pharmaceutical Compositions

IL-1 (e.g., IL-1β) binding antibodies and antibody fragments for useaccording to the present invention can be formulated in compositions,especially pharmaceutical compositions, for use in the methods herein.Such compositions comprise a therapeutically or prophylacticallyeffective amount of an IL-1β binding antibody or antibody fragment ofthe invention in admixture with a suitable carrier, e.g., apharmaceutically acceptable agent. Typically, IL-1β binding antibodiesand antibody fragments of the invention are sufficiently purified foradministration to an animal before formulation in a pharmaceuticalcomposition.

Pharmaceutically acceptable agents include carriers, excipients,diluents, antioxidants, preservatives, coloring, flavoring and dilutingagents, emulsifying agents, suspending agents, solvents, fillers,bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents,wetting agents, complexing agents, buffering agents, antimicrobials, andsurfactants.

Neutral buffered saline or saline mixed with albumin are exemplaryappropriate carriers. The pharmaceutical compositions can includeantioxidants such as ascorbic acid; low molecular weight polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as Tween, pluronics, or polyethylene glycol (PEG). Alsoby way of example, suitable tonicity enhancing agents include alkalimetal halides (preferably sodium or potassium chloride), mannitol,sorbitol, and the like. Suitable preservatives include benzalkoniumchloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid and the like. Hydrogen peroxide also can beused as preservative. Suitable cosolvents include glycerin, propyleneglycol, and PEG. Suitable complexing agents include caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxy-propyl-beta-cyclodextrin. Suitable surfactants or wetting agentsinclude sorbitan esters, polysorbates such as polysorbate 80,tromethamine, lecithin, cholesterol, tyloxapal, and the like. Thebuffers can be conventional buffers such as acetate, borate, citrate,phosphate, bicarbonate, or Tris-HCl. Acetate buffer may be about pH4-5.5, and Tris buffer can be about pH 7-8.5. Additional pharmaceuticalagents are set forth in Remington's Pharmaceutical Sciences, 18thEdition, A. R. Gennaro, ed., Mack Publishing Company, 1990.

The composition can be in liquid form or in a lyophilized orfreeze-dried form and may include one or more lyoprotectants,excipients, surfactants, high molecular weight structural additivesand/or bulking agents (see for example U.S. Pat. Nos. 6,685,940,6,566,329, and 6,372,716). In one embodiment, a lyoprotectant isincluded, which is a non-reducing sugar such as sucrose, lactose ortrehalose. The amount of lyoprotectant generally included is such that,upon reconstitution, the resulting formulation will be isotonic,although hypertonic or slightly hypotonic formulations also may besuitable. In addition, the amount of lyoprotectant should be sufficientto prevent an unacceptable amount of degradation and/or aggregation ofthe protein upon lyophilization. Exemplary lyoprotectant concentrationsfor sugars (e.g., sucrose, lactose, trehalose) in the pre-lyophilizedformulation are from about 10 mM to about 400 mM. In another embodiment,a surfactant is included, such as for example, nonionic surfactants andionic surfactants such as polysorbates (e.g. polysorbate 20, polysorbate80); poloxamers (e.g. poloxamer 188); poly (ethylene glycol) phenylethers (e.g. Triton); sodium dodecyl sulfate (SDS); sodium laurelsulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, orstearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearam idopropyl-betaine(e.g. lauroam idopropyl); myristarnidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl ofeyl-taurate; and the MONAQUAT™. series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g. Pluronics, PF68 etc). Exemplaryamounts of surfactant that may be present in the pre-lyophilizedformulation are from about 0.001-0.5%. High molecular weight structuraladditives (e.g. fillers, binders) may include for example, acacia,albumin, alginic acid, calcium phosphate (dibasic), cellulose,carboxymethylcellulose, carboxymethylcellulose sodium,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, microcrystalline cellulose, dextran,dextrin, dextrates, sucrose, tylose, pregelatinized starch, calciumsulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose,disodium hydrogen phosphate, disodium phosphate, disodium pyrosulfite,polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose,compressible sugar, magnesium aluminum silicate, maltodextrin,polyethylene oxide, polymethacrylates, povidone, sodium alginate,tragacanth microcrystalline cellulose, starch, and zein. Exemplaryconcentrations of high molecular weight structural additives are from0.1% to 10% by weight. In other embodiments, a bulking agent (e.g.,mannitol, glycine) may be included.

Compositions can be suitable for parenteral administration. Exemplarycompositions are suitable for injection or infusion into an animal byany route available to the skilled worker, such as intraarticular,subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral(intraparenchymal), intracerebroventricular, intramuscular, intraocular,intraarterial, intralesional, intrarectal, transdermal, oral, andinhaled routes. A parenteral formulation typically will be a sterile,pyrogen-free, isotonic aqueous solution, optionally containingpharmaceutically acceptable preservatives.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringers'dextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers, such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, anti-microbials, antioxidants, chelating agents, inertgases and the like. See generally, Remington's Pharmaceutical Science,16th Ed., Mack Eds., 1980, which is incorporated herein by reference.

Pharmaceutical compositions described herein can be formulated forcontrolled or sustained delivery in a manner that provides localconcentration of the product (e.g., bolus, depot effect) sustainedrelease and/or increased stability or half-life in a particular localenvironment. The invention contemplates that in certain embodiments suchcompositions may include a significantly larger amount of antibody orfragment in the initial deposit, while the effective amount of antibodyor fragment actually released and available at any point in time for isin accordance with the disclosure herein an amount much lower than theinitial deposit. The compositions can include the formulation of IL-1βbinding antibodies, antibody fragments, nucleic acids, or vectors of theinvention with particulate preparations of polymeric compounds such aspolylactic acid, polyglycolic acid, etc., as well as agents such as abiodegradable matrix, injectable microspheres, microcapsular particles,microcapsules, bioerodible particles beads, liposomes, and implantabledelivery devices that provide for the controlled or sustained release ofthe active agent which then can be delivered as a depot injection.Techniques for formulating such sustained- or controlled-delivery meansare known and a variety of polymers have been developed and used for thecontrolled release and delivery of drugs. Such polymers are typicallybiodegradable and biocompatible. Polymer hydrogels, including thoseformed by complexation of enantiomeric polymer or polypeptide segments,and hydrogels with temperature or pH sensitive properties, may bedesirable for providing drug depot effect because of the mild andaqueous conditions involved in trapping bioactive protein agents (e.g.,antibodies). See, for example, the description of controlled releaseporous polymeric microparticles for the delivery of pharmaceuticalcompositions in PCT Application Publication WO 93/15722.

Suitable materials for this purpose include polylactides (see, e.g.,U.S. Pat. No. 3,773,919), polymers of poly-(a-hydroxycarboxylic acids),such as poly-D-(−)-3-hydroxybutyric acid (EP 133,988A), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22: 547-556 (1983)), poly (2-hydroxyethyl-methacrylate) (Langer et al.,J. Biomed. Mater. Res., 15: 167-277 (1981), and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate, or poly-D(−)-3-hydroxybutyricacid. Other biodegradable polymers include poly(lactones),poly(acetals), poly(orthoesters), and poly(orthocarbonates).Sustained-release compositions also may include liposomes, which can beprepared by any of several methods known in the art (see, e.g., Eppsteinet al., Proc. Natl. Acad. Sci. USA, 82: 3688-92 (1985)). The carrieritself, or its degradation products, should be nontoxic in the targettissue and should not further aggravate the condition. This can bedetermined by routine screening in animal models of the target disorderor, if such models are unavailable, in normal animals.

Microencapsulation of recombinant proteins for sustained release hasbeen performed successfully with human growth hormone (rhGH),interferon- (rhIFN-), interleukin-2, and MN rgp120. Johnson et al., Nat.Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223 (1993); Horaet al., Bio/Technologv. 8:755-758 (1990); Cleland, “Design andProduction of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design: The Subunit andAdjuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.No. 5,654,010. The sustained-release formulations of these proteins weredeveloped using poly-lactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be depending on its molecular weight and composition. Lewis,“Controlled release of bioactive agents from lactide/glycolide polymer,”in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as DrugDelivery Systems (Marcel Dekker: New York, 1990), pp. 1-41. Additionalexamples of sustained release compositions include, for example, EP58,481A, U.S. Pat. No. 3,887,699, EP 158,277A, Canadian Patent No.1176565, U. Sidman et al., Biopolymers 22, 547 [1983], R. Langer et al.,Chem. Tech. 12, 98 [1982], Sinha et al., J. Control. Release 90, 261[2003], Zhu et al., Nat. Biotechnol. 18, 24 [2000], and Dai et al.,Colloids Surf B Biointerfaces 41, 117 [2005].

Bioadhesive polymers are also contemplated for use in or withcompositions of the present invention. Bioadhesives are synthetic andnaturally occurring materials able to adhere to biological substratesfor extended time periods. For example, Carbopol and polycarbophil areboth synthetic cross-linked derivatives of poly(acrylic acid).Bioadhesive delivery systems based on naturally occurring substancesinclude for example hyaluronic acid, also known as hyaluronan.Hyaluronic acid is a naturally occurring mucopolysaccharide consistingof residues of D-glucuronic and N-acetyl-D-glucosamine. Hyaluronic acidis found in the extracellular tissue matrix of vertebrates, including inconnective tissues, as well as in synovial fluid and in the vitreous andaqueous humour of the eye. Esterified derivatives of hyaluronic acidhave been used to produce microspheres for use in delivery that arebiocompatible and biodegrable (see for example, Cortivo et al.,Biomaterials (1991) 12:727-730; European Publication No. 517,565;International Publication No. WO 96/29998; Ilium et al., J. ControlledRel. (1994) 29:133-141). Exemplary hyaluronic acid containingcompositions of the present invention comprise a hyaluronic acid esterpolymer in an amount of approximately 0.1% to about 40% (w/w) of anIL-1β binding antibody or fragment to hyaluronic acid polymer.

Both biodegradable and non-biodegradable polymeric matrices can be usedto deliver compositions in accordance with the invention, and suchpolymeric matrices may comprise natural or synthetic polymers.Biodegradable matrices are preferred. The period of time over whichrelease occurs is based on selection of the polymer. Typically, releaseover a period ranging from between a few hours and three to twelvemonths is most desirable. Exemplary synthetic polymers which can be usedto form the biodegradable delivery system include: polymers of lacticacid and glycolic acid, polyam ides, polycarbonates, polyalkylenes,polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates,polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinylhalides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyanhydrides, polyurethanes and co-polymers thereof, poly(butic acid),poly(valeric acid), alkyl cellulose, hydroxyalkyl celluloses, celluloseethers, cellulose esters, nitro celluloses, polymers of acrylic andmethacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetatebutyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, cellulose sulphate sodium salt, poly(methylmethacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), poly(octadecyl acrylate), polyethylene, polypropylene,poly(ethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinylchloride, polystyrene and polyvinylpyrrolidone. Exemplary naturalpolymers include alginate and other polysaccharides including dextranand cellulose, collagen, chemical derivatives thereof (substitutions,additions of chemical groups, for example, alkyl, alkylene,hydroxylations, oxidations, and other modifications routinely made bythose skilled in the art), albumin and other hydrophilic proteins, zeinand other prolamines and hydrophobic proteins, copolymers and mixturesthereof. In general, these materials degrade either by enzymatichydrolysis or exposure to water in vivo, by surface or bulk erosion. Thepolymer optionally is in the form of a hydrogel (see for example WO04/009664, WO 05/087201, Sawhney, et al., Macromolecules, 1993, 26,581-587) that can absorb up to about 90% of its weight in water andfurther, optionally is cross-linked with multi-valent ions or otherpolymers.

Delivery systems also include non-polymer systems that are lipidsincluding sterols such as cholesterol, cholesterol esters and fattyacids or neutral fats such as mono- di- and tri-glycerides; hydrogelrelease systems; silastic systems; peptide based systems; wax coatings;compressed tablets using conventional binders and excipients; partiallyfused implants; and the like. Specific examples include, but are notlimited to: (a) erosional systems in which the product is contained in aform within a matrix such as those described in U.S. Pat. Nos.4,452,775, 4,675,189 and 5,736,152 and (b) diffusional systems in whicha product permeates at a controlled rate from a polymer such asdescribed in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686.Liposomes containing the product may be prepared by methods knownmethods, such as for example (DE 3,218,121; Epstein et al., Proc. Natl.Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP143,949; EP 142,641; Japanese patent application 83-118008; U.S. Pat.Nos. 4,485,045 and 4,544,545; and EP 102,324).

A pharmaceutical composition comprising an IL-1β binding antibody orfragment can be formulated for inhalation, such as for example, as a drypowder. Inhalation solutions also can be formulated in a liquefiedpropellant for aerosol delivery. In yet another formulation, solutionsmay be nebulized. Additional pharmaceutical composition for pulmonaryadministration include, those described, for example, in PCT ApplicationPublication WO 94/20069, which discloses pulmonary delivery ofchemically modified proteins. For pulmonary delivery, the particle sizeshould be suitable for delivery to the distal lung. For example, theparticle size can be from 1 μm to 5 μm; however, larger particles may beused, for example, if each particle is fairly porous.

Certain formulations containing IL-1β binding antibodies or antibodyfragments can be administered orally. Formulations administered in thisfashion can be formulated with or without those carriers customarilyused in the compounding of solid dosage forms such as tablets andcapsules. For example, a capsule can be designed to release the activeportion of the formulation at the point in the gastrointestinal tractwhen bioavailability is maximized and pre-systemic degradation isminimized. Additional agents can be included to facilitate absorption ofa selective binding agent. Diluents, flavorings, low melting pointwaxes, vegetable oils, lubricants, suspending agents, tabletdisintegrating agents, and binders also can be employed.

Another preparation can involve an effective quantity of an IL-16binding antibody or fragment in a mixture with non-toxic excipientswhich are suitable for the manufacture of tablets. By dissolving thetablets in sterile water, or another appropriate vehicle, solutions canbe prepared in unit dose form. Suitable excipients include, but are notlimited to, inert diluents, such as calcium carbonate, sodium carbonateor bicarbonate, lactose, or calcium phosphate; or binding agents, suchas starch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

Suitable and/or preferred pharmaceutical formulations can be determinedin view of the present disclosure and general knowledge of formulationtechnology, depending upon the intended route of administration,delivery format, and desired dosage. Regardless of the manner ofadministration, an effective dose can be calculated according to patientbody weight, body surface area, or organ size. Further refinement of thecalculations for determining the appropriate dosage for treatmentinvolving each of the formulations described herein are routinely madein the art and is within the ambit of tasks routinely performed in theart. Appropriate dosages can be ascertained through use of appropriatedose-response data.

Additional formulations will be evident in light of the presentdisclosure, including formulations involving IL-1β binding antibodiesand fragments in combination with one or more other therapeutic agents.For example, in some formulations, an IL-1β binding antibody, antibodyfragment, nucleic acid, or vector of the invention is formulated with asecond inhibitor of an IL-1 signaling pathway Representative secondinhibitors include, but are not limited to, antibodies, antibodyfragments, peptides, polypeptides, compounds, nucleic acids, vectors andpharmaceutical compositions, such as, for example, those described inU.S. Pat. No. 6,899,878, US 2003022869, US 20060094663, US 20050186615,US 20030166069, WO/04022718, WO/05084696, WO/05019259. For example, acomposition may comprise an IL-1β binding antibody, antibody fragment,nucleic acid, or vector of the invention in combination with anotherIL-1β binding antibody, fragment, or a nucleic acid or vector encodingsuch an antibody or fragment.

The pharmaceutical compositions can comprise IL-1β binding antibodies orfragments in combination with other active agents. Such combinations arethose useful for their intended purpose. The combinations which are partof this invention can be IL-1β antibodies and fragments, such as forexample those described herein, and at least one additional agent.Examples of active agents that may be used in combination set forthbelow are illustrative for purposes and not intended to be limited. Thecombination can also include more than one additional agent, e.g., twoor three additional agents if the combination is such that the formedcomposition can perform its intended function.

The invention further contemplates that pharmaceutical compositionscomprising one or more other active agents may be administeredseparately from the IL-1β binding antibodies or fragments, and suchseparate administrations may be performed at the same point or differentpoints in time, such as for example the same or different days.Administration of the other active agents may be according to standardmedical practices known in the art, or the administration may bemodified (e.g., longer intervals, smaller dosages, delayed initiation)when used in conjunction with administration of IL-1β binding antibodiesor fragments, such as disclosed herein.

Active agents or combinations with the present antibodies or fragmentsinclude indomethacin, non-steroidal anti-inflammatory drugs (NSAIDs)such as aspirin, ibuprofen, and other propionic acid derivatives(alminoprofen, benoxaprofen, bucloxic acid, carprofen, fenbufen,fenoprofen, fluprofen, flurbiprofen, indoprofen, ketoprofen, miroprofen,naproxen, oxaprozin, pirprofen, pranoprofen, suprofen, tiaprofenic acid,and tioxaprofen), acetic acid derivatives (indomethacin, acemetacin,alclofenac, clidanac, diclofenac, fenclofenac, fenclozic acid,fentiazac, fuirofenac, ibufenac, isoxepac, oxpinac, sulindac, tiopinac,tolmetin, zidometacin, and zomepirac), fenamic acid derivatives(flufenamic acid, meclofenamic acid, mefenamic acid, niflumic acid andtolfenamic acid), biphenylcarboxylic acid derivatives (diflunisal andflufenisal), oxicams (isoxicam, piroxicam, sudoxicam and tenoxican),salicylates (acetyl salicylic acid, sulfasalazine) and the pyrazolones(apazone, bezpiperylon, feprazone, mofebutazone, oxyphenbutazone,phenylbutazone). Other combinations include cyclooxygenase-2 (COX-2)inhibitors, aquaretics, oral glucocorticoids, intra-articularglucocorticoids, colchicine, xanthine-oxidase inhibitors, allopurinol,uricosuric agents, sulfinpyrazone, febuxostat, probenecid, fenofibrate,benemid, angiotensin II receptor antagonists, losartan, thiazides,PEG-uricase, sodium bicarbonate, ethylenediaminetetraacetic acid. Otheractive agents for combination include steroids such as prednisolone,prednisone, methylprednisolone, betamethasone, dexamethasone, orhydrocortisone. Such a combination may be especially advantageous, sinceone or more side-effects of the steroid can be reduced or eveneliminated by tapering the steroid dose required when treating patientsin combination with the present antibodies and fragments.

It is further contemplated that an anti-IL-1β antibody or fragmentadministered to a subject in accordance with the invention may beadministered in combination with treatment with at least one additionalactive agent, such as for example any of the aforementioned activeagents. In one embodiment, treatment with the at least one active agentis maintained. In another embodiment, treatment with the at least oneactive agent is reduced or discontinued (e.g., when the subject isstable) during the course of IL-1β antibody treatment (e.g., with theanti-IL-1β antibody or fragment maintained at a constant dosing regimen.In another embodiment, treatment with the at least one active agent isreduced or discontinued (e.g., when the subject is stable), andtreatment with the anti-IL-1β antibody or fragment is reduced (e.g.,lower dose, less frequent dosing, shorter treatment regimen). In anotherembodiment, treatment with the at least one active agent is is reducedor discontinued (e.g., when the subject is stable), and treatment withthe anti-IL-1β antibody or fragment is increased (e.g., higher dose,more frequent dosing, longer treatment regimen). In yet anotherembodiment, treatment with the at least one active agent is maintainedand treatment with the anti-IL-1β antibody or fragment is reduced ordiscontinued (e.g., lower dose, less frequent dosing, shorter treatmentregimen). In yet another embodiment, treatment with the at least oneactive agent and treatment with the anti-IL-1β antibody or fragment arereduced or discontinued (e.g., lower dose, less frequent dosing, shortertreatment regimen)

The pharmaceutical compositions used in the invention may include atherapeutically effective amount or a prophylactically effective amountof the IL-1β binding antibodies or fragments. A therapeuticallyeffective amount refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the antibody or antibody portion mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the antibody or antibodyportion to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody or antibody portion are outweighedby the therapeutically beneficial effects. A prophylactically effectiveamount refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired prophylactic result.

A therapeutically or prophylactically effective amount of apharmaceutical composition comprising an IL-1β binding antibody orfragment will depend, for example, upon the therapeutic objectives suchas the indication for which the composition is being used, the route ofadministration, and the condition of the subject. Pharmaceuticalcompositions are administered in a therapeutically or prophylacticallyeffective amount to treat an IL-1 related condition.

Methods of Use

Anti-IL-1β antibodies as provided herein may be used for the treatmentand/or prevention of gout in a subject. Such methods may be used totreat a mammalian subject (e.g., human) suffering from gout or toprevent occurrence of the same in an at risk subject.

The terms “prevention”, “prevent”, “preventing”, “suppression”,“suppress”, “suppressing”, “inhibit” and “inhibition” as used hereinrefer to a course of action (such as administering a compound orpharmaceutical composition) initiated in a manner (e.g., prior to theonset of a clinical symptom of a disease state or condition) so as toprevent, suppress or reduce, either temporarily or permanently, theonset of a clinical manifestation of the disease state or condition.Such preventing, suppressing or reducing need not be absolute to beuseful.

The terms “treatment”, “treat” and “treating” as used herein refers acourse of action (such as administering a compound or pharmaceuticalcomposition) initiated after the onset of a clinical symptom of adisease state or condition so as to eliminate, reduce, suppress orameliorate, either temporarily or permanently, a clinical manifestationor progression of the disease state or condition. Such treating need notbe absolute to be useful.

The term “in need of treatment” as used herein refers to a judgment madeby a caregiver that a patient requires or will benefit from treatment.This judgment is made based on a variety of factors that are in therealm of a caregiver's expertise, but that includes the knowledge thatthe patient is ill, or will be ill, as the result of a condition that istreatable by a method or compound of the disclosure.

The term “in need of prevention” as used herein refers to a judgmentmade by a caregiver that a patient requires or will benefit fromprevention. This judgment is made based on a variety of factors that arein the realm of a caregiver's expertise, but that includes the knowledgethat the patient will be ill or may become ill, as the result of acondition that is preventable by a method or compound of the disclosure.

The term “therapeutically effective amount” as used herein refers to anamount of a compound (e.g., antibody), either alone or as a part of apharmaceutical composition, that is capable of having any detectable,positive effect on any symptom, aspect, or characteristics of a diseasestate or condition when administered to a patient (e.g., as one or moredoses). Such effect need not be absolute to be beneficial.

In one embodiment, the anti-IL-1β antibody or fragment is administeredto a subject with gout and the subject also receives at least one othermedically accepted treatment (e.g, medication, drug, therapeutic, activeagent) for the disease, condition or complication. In anotherembodiment, the at least one other medically accepted treatment for thedisease, condition or complication is reduced or discontinued (e.g.,when the subject is stable), while treatment with the anti-IL-1βantibody or fragment is maintained at a constant dosing regimen. Inanother embodiment, the at least one other medically accepted treatmentfor the disease, condition or complication is reduced or discontinued(e.g., when the subject is stable), and treatment with the anti-IL-1βantibody or fragment is reduced (e.g., lower dose, less frequent dosing,shorter treatment regimen). In another embodiment, the at least oneother medically accepted treatment for the disease, condition orcomplication is reduced or discontinued (e.g., when the subject isstable), and treatment with the anti-IL-1β antibody or fragment isincreased (e.g., higher dose, more frequent dosing, longer treatmentregimen). In yet another embodiment, the at least one other medicallyaccepted treatment for the disease, condition or complication ismaintained and treatment with the anti-IL-1β antibody or fragment isreduced or discontinued (e.g., lower dose, less frequent dosing, shortertreatment regimen). In yet another embodiment, the at least one othermedically accepted treatment for the disease, condition or complicationand treatment with the anti-IL-1β antibody or fragment are reduced ordiscontinued (e.g., lower dose, less frequent dosing, shorter treatmentregimen)

In preferred methods of treating or preventing gout, anti-IL-1β antibodyor fragment thereof is administered to the subject according to theaforementioned numbers of doses, amounts per dose and/or intervalsbetween dosing. Alternatively, the anti-IL-1β antibody or fragment maybe administered as one or more initial doses of the aforementionedamounts that are lower than one or more subsequent dose amounts. Byproviding the initial dose(s) in a lower amount, the effectivenessand/or tolerability of the treatment may be enhanced. For example, in anon-limiting embodiment of the invention, one or more initial doses(e.g., 1, 2, 3, 4, 5) of an amount of antibody or fragment ≤1 mg/kg(e.g., ≤0.9 mg/kg, ≤0.8 mg/kg, ≤0.7 mg/kg, ≤0.6 mg/kg, ≤0.5 mg/kg, ≤0.4mg/kg, ≤0.3 mg/kg, ≤0.2 mg/kg, ≤0.1 mg/kg, ≤0.05 mg/kg, ≤0.03 mg/kg,≤0.01 mg/kg) may be administered, followed by one or more subsequentdoses in an amount greater than the initial dose(s) (e.g., ≥0.01 mg/kg,≥0.03 mg/kg, ≥0.1 mg/kg, ≥0.3 mg/kg ≥0.5 mg/kg, ≥0.6 mg/kg, ≥0.7 mg/kg,≥0.8 mg/kg, ≥0.9 mg/kg, ≥1.0 mg/kg, ≥1.5 mg/kg, ≥2 mg/kg, ≥2.5 mg/kg, ≥3mg/kg, ≥3.5 mg/kg, ≥4 mg/kg, ≥4.5 mg/kg, ≥5 mg/kg). The inventioncontemplates that each dose of antibody or fragment may be administeredat one or more sites.

Methods of treating or preventing a disease or condition in accordancewith the present invention may use a pre-determined or “routine”schedule for administration of the antibody or fragment. As used hereina routine schedule refers to a predetermined designated period of timebetween dose administrations. The routine schedule may encompass periodsof time which are identical or which differ in length, as long as theschedule is predetermined. Any particular combination would be coveredby the routine schedule as long as it is determined ahead of time thatthe appropriate schedule involves administration on a certain day.

The invention further contemplates that IL-1β antibodies or fragmentsused in accordance with the methods provided herein, may be administeredin conjunction with more traditional treatment methods andpharmaceutical compositions (e.g., active agents). Such compositions mayinclude for example, nonsteroidal anti-inflammatory drugs (NSAIDs)corticosteroids, adrenocorticotropic hormone, and colchicines. Incertain embodiments, the antibodies and fragments used in accordancewith the invention may prevent or delay the need for additionaltreatment methods or pharmaceutical compositions. In other embodiments,the antibodies or fragments may reduce the amount, frequency or durationof additional treatment methods or pharmaceutical compositions.

Alternatively, methods of treating or preventing a disease or conditionin accordance with the present invention may use a schedule foradministration of the antibody or fragment that is based upon thepresence of disease symptoms and/or changes in any of the assessmentsherein as a means to determine when to administer one or more subsequentdoses. Similar, this approach may be used as a means to determinewhether a subsequent dose should be increased or decreased, based uponthe effect of a previous dose.

Diagnosis of such diseases or conditions in patients, or alternativelythe risk for developing such diseases or conditions may be according tostandard medical practices known in art. Following administration of ananti-IL-1β antibodies or fragment, clinical assessments for a treatmentor preventative effect on gout are well known in the art and may be usedas a means to monitor the effectiveness of methods of the invention. Forexample, response to treatment of gout may be assessed based on aclinical assessment of the acute gout episode that includes aphysician's assessment assessing redness, tenderness, and swelling (noneof which are attributable to other causes), a physician's globalassessment, a subject pain self-assessment, a patient's globalassessment, and/or a HAQ. In one embodiment, efficacy of treatment isassessed by a reduction in joint pain of at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, or about 100%. In anotherembodiment, the reduction in join pain occurs in less than about 48hours, less than about 36 hours, less than about 24 hours. The clinicalassessment of acute gout may include one or more of the followingcomponents:

Physician's Assessments:

-   -   Physician's Global Assessment (10-point analog scale)    -   Physician's assessment of erythema (10-point analog scale)    -   Physician's assessment of heat (10-point analog scale)    -   Physician's assessment of swelling (10-point analog scale)

Subject's Assessments:

-   -   Patient's Global Assessment (10-point analog scale)    -   Pain at rest (10-point analog scale)    -   Pain on weight-bearing/movement (10-point analog scale)    -   Health Assessment Questionnaire (HAQ)

One or more secondary endpoints, such as for example C-reactive protein(CRP) levels and/or erythrocyte sedimentation rate (ESR) also may bedetermined in order to assess efficacy of the treatment. A decrease inCRP levels of ≥0.2, ≥0.4, ≥0.6, ≥0.8, ≥1.0, ≥1.4, ≥1.8, ≥2.2, ≥2.6, ≥3.0mg/L; alternatively a decrease of >20%, >30%,≥40%, >50%, >60%, >70%, >80%, >90%, >95% from pre-treatment levels isindicative of therapeutic effect. A decrease in ESRof >20%, >30%, >40%, >50%, >60%, >70%, ≥80%, >90%, >95%, >98% frompre-treatment levels is indicative of therapeutic effect. The disclosureprovides a method of treating gout in a subject (e.g., human subject),the method comprising administering (e.g., in a therapeuticallyeffective amount) an anti-IL-1β antibody or fragment thereof to thesubject, wherein the dose of the antibody or fragment is sufficient toachieve at least a 50% reduction in joint pain and at least a 20%decrease in CRP levels, at least a 30% decrease in CRP levels, at leasta 40% decrease in CRP levels, at least a 50% decrease in CRP levels, atleast a 60% decrease in CRP levels, at least a 70% decrease in CRPlevels, at least a 80% decrease in CRP levels, and/or at least a 90%decrease in CRP levels. In a preferred embodiment, the dose of theantibody or fragment is sufficient to achieve at least a 50% reductionin joint pain and at least a 20% decrease in ESR, at least a 40%decrease in ESR, at least a 50% decrease in ESR, at least a 60% decreasein ESR, at least a 70% decrease in ESR, at least a 80% decrease in ESR,and/or at least a 90% decrease in ESR.

The disclosure also provides a method of treating gout in a subject(e.g., human subject), the method comprising administering (e.g., in atherapeutically effective amount) an anti-IL-1β antibody or fragmentthereof to the subject, wherein the dose of the antibody or fragment issufficient to achieve at least a 50% reduction in joint pain, at least a20% decrease in CRP levels and at least a 20% decrease in ESR. In oneembodiment, the dose of the antibody or fragment is sufficient toachieve at least a 50% reduction in joint pain, at least a 30% decreasein CRP levels and a 30% decrease in ESR. In another embodiment, the doseof the antibody or fragment is sufficient to achieve at least a 50%reduction in joint pain, at least a 40% decrease in CRP levels and a 40%decrease in ESR. In another embodiment, the dose of the anti-IL-1βantibody or fragment is sufficient to achieve at least a 60% reductionin joint pain, at least a 20% decrease in CRP levels and at least a 20%decrease in ESR. In another embodiment, the dose of the anti-IL-1βantibody or fragment is sufficient to achieve at least a 60% reductionin joint pain, at least a 40% decrease in CRP levels and at least a 40%decrease in ESR. In another embodiment, the dose of the anti-IL-1βantibody or fragment is sufficient to achieve at least a 60% reductionin joint pain, at least a 50% decrease in CRP levels and at least a 50%decrease in ESR. In yet another embodiment, the dose of the anti-IL-1βantibody or fragment is sufficient to achieve at least a 70% reductionin joint pain, at least a 20% decrease in CRP levels and at least a 20%decrease in ESR. In another embodiment, the dose of the anti-IL-1βantibody or fragment is sufficient to achieve at least a 70% reductionin joint pain, at least a 40% decrease in CRP levels and at least a 40%decrease in ESR. In another embodiment, the dose of the anti-IL-1βantibody or fragment is sufficient to achieve at least a 70% reductionin joint pain, at least a 50% decrease in CRP levels and at least a 50%decrease in ESR. In one embodiment, CRP levels may be measured by anultra-sensitive CRP ELISA test. In another embodiment, ESR may bemeasured by a Westergren ESR test method.

ADDITIONAL EMBODIMENTS

-   -   1. A method of treating gout in a subject, the method comprising        administering an anti-IL-1β antibody or fragment thereof to the        subject.    -   2. The method of embodiment 1, wherein the gout is chronic gout.    -   3. The method of embodiment 1, wherein the gout is acute gout.    -   4. The method of embodiments 1-3, wherein the antibody or        antibody fragment binds to human IL-1β with a dissociation        constant of about 10 nM or less.    -   5. The method of embodiments 4, wherein the antibody or antibody        fragment binds to human IL-1β with a dissociation constant of        about 1 nM or less.    -   6. The method of embodiments 5, wherein the antibody or antibody        fragment binds to human IL-1β with a dissociation constant of        about 500 pM or less.    -   7. The method of embodiments 6, wherein the antibody or antibody        fragment binds to human IL-1β with a dissociation constant of        about 250 pM or less.    -   8. The method of embodiment 7, wherein the antibody or antibody        fragment binds to human IL-1β with a dissociation constant of        about 10 pM or less.    -   9. The method of embodiment 8, wherein the antibody or antibody        fragment binds to human IL-1β with a dissociation constant of        about 1 pM or less.    -   10. The method of embodiment 9, wherein the antibody or antibody        fragment binds to human IL-1β with a dissociation constant of        about 0.5 pM or less.    -   11. The method of embodiment 10, wherein the antibody or        antibody fragment binds to human IL-1β with a dissociation        constant of about 0.3 pM or less.    -   12. The method of embodiments 1-11, wherein the anti-IL-1β        antibody or antibody fragment is a neutralizing antibody.    -   13. The method of embodiments 1-11, wherein the anti-IL-1β        antibody or antibody fragment binds to an IL-1β epitope such        that the bound antibody or fragment substantially permits the        binding of IL-1β to IL-1 receptor I (IL-1RI).    -   14. The method of embodiments 1-11, wherein the antibody or        antibody fragment does not detectably bind to IL-1α, IL-1R or        IL-1Ra.    -   15. The method of embodiments 1-11, wherein the antibody or        antibody fragment binds to an epitope contained in the sequence

(SEQ ID NO. 1) ESVDPKNYPKKKMEKRFVFNKIE.

-   -   16. The method of embodiments 1-11, wherein the antibody or        fragment thereof competes with the binding of an antibody having        the light chain variable region of SEQ ID NO:5 and the heavy        chain variable region of SEQ ID NO:6    -   17. The method of embodiments 1-11, wherein the antibody or        antibody fragment binds to an epitope incorporating Glu64 of        IL-1β.    -   18. The method of embodiments 1-11, wherein the antibody or        antibody fragment binds to amino acids 1-34 of the N terminus of        IL-1β.    -   19. The method of embodiments 1-11, wherein the antibody or        antibody fragment is human engineered or humanized.    -   20. The method of embodiments 1-11, wherein the antibody or        antibody fragment is human.    -   21. The method of embodiments 1-20, wherein the antibody or        antibody fragment is administered in one or more doses of 3        mg/kg or less of antibody or fragment.    -   22. The method of embodiment 21, wherein the antibody or        antibody fragment is administered in one or more doses of 1        mg/kg or less of antibody or fragment.    -   23. The method of embodiment 22, wherein the antibody or        antibody fragment is administered in one or more doses of 0.3        mg/kg or less of antibody or fragment.    -   24. The method of embodiment 23, wherein the antibody or        antibody fragment is administered in one or more doses of 0.1        mg/kg or less of antibody or fragment.    -   25. The method of embodiment 24, wherein the antibody or        antibody fragment is administered in one or more doses of 0.03        mg/kg or less of antibody or fragment.    -   26. The method of embodiment 25, wherein the antibody or        antibody fragment is administered in one or more doses of 0.01        mg/kg or less of antibody or fragment.    -   27. The method of embodiment 26, wherein the antibody or        antibody fragment is administered in one or more doses of 0.003        mg/kg or less of antibody or fragment.    -   28. The method of embodiment 27, wherein the antibody or        antibody fragment is administered in one or more doses of 0.001        mg/kg or less of antibody or fragment.    -   29. The method of embodiments 21-28, wherein the one or more        doses are at least 0.001 mg/kg of antibody or fragment.    -   30. The method of embodiments 21-26, wherein the one or more        doses are at least 0.01 mg/kg of antibody or fragment.    -   31. The method of embodiments 1-20, wherein the antibody or        antibody fragment is administered in one or more doses of 0.001        mg/kg to 1 mg/kg.    -   32. The method of embodiment 31, wherein the antibody or        antibody fragment is administered in one or more doses of 0.001        mg/kg to 0.3 mg/kg.    -   33. The method of embodiment 31, wherein the antibody or        antibody fragment is administered in one or more doses of 0.003        mg/kg to 1 mg/kg.    -   34. The method of embodiment 33, wherein the antibody or        antibody fragment is administered in one or more doses of 0.003        mg/kg to 0.3 mg/kg.    -   35. The method of embodiments 1-20, wherein the antibody or        fragment is administered as a fixed dose, independent of a dose        per subject weight ratio.    -   36. The method of embodiment 35, wherein the antibody or        fragment is administered in one or more doses of 500 mg or less        of antibody or fragment.    -   37. The method of embodiment 36, wherein the antibody or        fragment is administered in one or more doses of 250 mg or less        of antibody or fragment.    -   38. The method of embodiment 37, wherein the antibody or        fragment is administered in one or more doses of 100 mg or less        of antibody or fragment.    -   39. The method of embodiment 38, wherein the antibody or        fragment is administered in one or more doses of 25 mg or less        of antibody or fragment.    -   40. The method of embodiment 39, wherein the antibody or        fragment is administered in one or more doses of 10 mg or less        of antibody or fragment.    -   41. The method of embodiment 40, wherein the antibody or        fragment is administered in one or more doses of 1.0 mg or less        of antibody or fragment.    -   42. The method of embodiments 35-41, wherein the antibody or        fragment is administered in one or more doses of at least 0.1 mg        of antibody or fragment.    -   43. The method of embodiment 42, wherein the antibody or        fragment is administered in one or more doses of at least 1.0 mg        of antibody or fragment.    -   44. The method of embodiments 35-40, wherein the antibody or        fragment is administered in one or more doses of at least 10 mg        of antibody or fragment.    -   45. The method of embodiments 1-44, wherein the anti-IL-1β        antibody or fragment is administered by subcutaneous,        intravenous or intramuscular injection    -   46. The method of embodiments 1-45, wherein administration of an        initial dose of the antibody or antibody fragment is followed by        the administration of one or more subsequent doses.    -   47. The method of embodiments 1-45, wherein administration of an        initial dose of the antibody or antibody fragment is followed by        the administration of one or more subsequent doses, and wherein        said one or more subsequent doses are in an amount that is        approximately the same or less than the initial dose.    -   48. The method of embodiments 1-45, wherein administration of an        initial dose of the antibody or antibody fragment is followed by        the administration of one or more subsequent doses, and wherein        at least one of the subsequent doses is in an amount that is        more than the initial dose.    -   49. The method of embodiments 1-48, wherein the dose of the        antibody or fragment is sufficient to achieve at least a 50%        reduction in joint pain.    -   50. The method of embodiment 49, wherein the dose of the        antibody or fragment is sufficient to achieve at least a 60%        reduction in joint pain.    -   51. The method of embodiment 50, wherein the dose of the        antibody or fragment is sufficient to achieve at least a 70%        reduction in joint pain.    -   52. The method of embodiment 51, wherein the dose of the        antibody or fragment is sufficient to achieve at least a 80%        reduction in joint pain.    -   53. The method of embodiment 52, wherein the dose of the        antibody or fragment is sufficient to achieve at least a 90%        reduction in joint pain.    -   54. The method of embodiment 52, wherein the dose of the        antibody or fragment is sufficient to achieve a 95% reduction in        joint pain.    -   55. The method of embodiments 1-54, wherein the dose of the        antibody or fragment is sufficient to achieve at least a 20%        decrease in CRP levels.    -   56. The method of embodiment 55, wherein the dose of the        antibody or fragment is sufficient to achieve at least a 30%        decrease in CRP levels.    -   57. The method of embodiment 56, wherein the dose of the        antibody or fragment is sufficient to achieve at least a 40%        decrease in CRP levels.    -   58. The method of embodiment 57, wherein the dose of the        antibody or fragment is sufficient to achieve at least a 50%        decrease in CRP levels.    -   59. The method of embodiment 58, wherein the dose of the        antibody or fragment is sufficient to achieve at least a 70%        decrease in CRP levels.    -   60. The method of embodiment 59, wherein the dose of the        antibody or fragment is sufficient to achieve at least a 90%        decrease in CRP levels.    -   61. The method of embodiments 1-54, wherein the dose of the        antibody or fragment is sufficient to achieve at least a 20%        decrease in ESR.    -   62. The method of embodiment 61, wherein the dose of the        antibody or fragment is sufficient to achieve at least a 40%        decrease in ESR.    -   63. The method of embodiment 62, wherein the dose of the        antibody or fragment is sufficient to achieve at least a 60%        decrease in ESR.    -   64. The method of embodiment 63, wherein the dose of the        antibody or fragment is sufficient to achieve at least a 70%        decrease in ESR.    -   65. The method of embodiment 64, wherein the dose of the        antibody or fragment is sufficient to achieve at least a 80%        decrease in ESR.    -   66. The method of embodiment 65, wherein the dose of the        antibody or fragment is sufficient to achieve at least a 90%        decrease in ESR.    -   67. The method of embodiments 1-54, wherein the dose of the        antibody or fragment is sufficient to achieve at least a 50%        reduction in joint pain, at least a 20% decrease in CRP and at        least a 20% decrease in ESR.    -   68. The method of embodiment 67, wherein the dose of the        antibody or fragment is sufficient to achieve at least a 30%        decrease in CRP and at least a 30% decrease in ESR.    -   69. The method of embodiment 68, wherein the dose of the        antibody or fragment is sufficient to achieve at least a 40%        decrease in CRP and at least a 40% decrease in ESR.    -   70. The method of embodiments 1-69, wherein said method is in        conjunction with at least one additional treatment method, said        additional treatment method comprising administering at least        one pharmaceutical composition comprising an active agent other        than an IL-1β antibody or fragment.    -   71. The method of embodiment 70, wherein said at least one        pharmaceutical composition comprising an active agent other than        an IL-1β antibody or fragment is selected from the group        consisting of a nonsteroidal anti-inflammatory drug (NSAID), a        corticosteroid, an adrenocorticotropic hormone, and a        colchicines.    -   72. The method of embodiments 1-71, wherein the antibody or        fragment thereof has a lower IC₅₀ than an IL-1β receptor        antagonist in a human whole blood IL-1β inhibition assay that        measures IL-1β induced production of IL-8.    -   73. The use of an anti-IL-1β antibody or fragment thereof which        has a lower IC₅₀ than an IL-1β receptor antagonist in a human        whole blood IL-1β inhibition assay that measures IL-1β induced        production of IL-8, in the manufacture of a composition for use        in the treatment of gout.    -   74. The method of embodiment 72 or the use according to        embodiment 73, wherein the IL-1β receptor antagonist is        anakinra.

EXAMPLES

The following examples are intended merely to further illustrate thepractice of the present invention, but should not be construed as in anyway limiting its scope. The disclosures of all patent and scientificliteratures cited within are hereby expressly incorporated in theirentirety by reference.

Example 1 Inhibition of IL-1β Using a High Affinity IL-1β Antibody in anIn Vitro Cell Based Assay, with IL-1 Induced Production of IL-8 as aRead-Out

The inhibitory effect of the IL-1β-specific antibody was compared to anon-antibody inhibitor of the IL-1 pathway, Kineret® (anakinra), whichis a recombinant IL-1 receptor antagonist (IL-1Ra). Fresh, heparinizedperipheral blood was collected from healthy donors. 180 μl of wholeblood was plated in a 96-well plate and incubated with variousconcentrations of the antibody AB7 (U.S. application Ser. No.11/472,813, WO 2007/002261) and 100 pM rhIL-1β. For Kineret®-treatedsamples, Kineret® and rhIL-1β were combined 1:1 prior to mixing withblood. Samples were incubated for 6 hours at 37° C. with 5% CO₂. Wholeblood cells were then lysed with 50 μl 2.5% Triton X-100. Theconcentration of interleukin-8 (IL-8) in cleared lysates was assayed byELISA (Quantikine human IL-8 ELISA kit, R&D Systems) according tomanufacturer's instructions. IL-8 concentrations in AB7 and Kineret®treated samples were compared to a control sample treated with anti-KLHcontrol. The results are depicted in FIG. 1 and summarized in Table 6.IC₅₀ is the concentration of antibody required to inhibit 50% of IL-8released by IL-1β stimulation.

TABLE 1 IC₅₀ (pM) AB7  1.9 pM Kineret ® 53.4 pM

These results demonstrate the in vitro potency of AB7, as measured byinhibition of IL-1β stimulated release of IL-8. The results showinggreater potency compared with Kineret® indicate that the antibody willhave IL-1β inhibitory efficacy in vivo.

Example 2 In Vivo Inhibition of the Biological Activity of Human IL-1βUsing IL-1β-Specific Antibodies, as Measured by the Impact on IL-1βStimulated Release of IL-6

To confirm the in vivo efficacy of AB7, its ability to block thebiological activity of human IL-1β was tested in mice. Details of theassay are described in Economides et al., Nature Med., 9: 47-52 (2003).Briefly, male C57/B16 mice (Jackson Laboratory Bar Harbor, Me.) wereinjected intraperitoneally with titrated doses of AB7, another IL-1βantibody, AB5, or a control antibody. Twenty-four hours after antibodyinjection, mice were injected subcutaneously with recombinant humanIL-1β (rhIL-1β) (from PeproTech Inc., Rocky Hill, N.J.) at a dose of 1pg/kg. Two hours post-rhIL-1β injection (peak IL-6 response time), micewere sacrificed, and blood was collected and processed for serum. SerumIL-6 levels were assayed by ELISA (BD Pharmingen, Franklin Lakes, N.J.)according to the manufacturer's protocol. Percent inhibition wascalculated from the ratio of IL-6 detected in experimental animal serumto IL-6 detected in control animal serum (multiplied by 100).

The results are set forth in FIG. 2A. The ability to inhibit the in vivoactivity of IL-1β was assessed as a function of IL-1β stimulated IL-6levels in serum. As illustrated by FIG. 2A, the AB7 and AB5 antibodieswere effective for inhibiting the in vivo activity of human IL-1β. Theseresults also show that a single injection of AB7 or AB5 can block thesystemic action to IL-1β stimulation and that such antibodies are usefulfor the inhibition of IL-1β activity in vivo.

A similar experiment was performed to further demonstrate the ability ofAB7 to neutralize mouse IL-1β in vivo, to support the use of thisantibody in mouse models of disease. It was determined that AB7 has anaffinity for human IL-1β that is ˜10,000 times greater than the affinityfor mouse IL-1β, and an in vitro potency in the D10.G4.1 assay that is˜1,000 times greater than that for mouse IL-1β. In the C57BL/6 mousemodel with IL-6 readout, the mice were injected with AB7 (3 or 300 ug)or PBS vehicle control i.p. 24 hours before a s.c. injection of human(FIG. 2B, panel A) or mouse (FIG. 2B, panel B) IL-1β (20 ng). Blood wasdrawn 2 hours later and serum samples were analyzed for IL-6 levels viaELISA. These data show maximum suppression of IL-6 levels (˜75%) inducedby human IL-1β at 3 μg (panel A), whereas submaximum suppression of IL-6levels (˜50%) induced by mouse IL-1β was demonstrated with 300 μg (panelB). These results are consistent with the observation of far greateraffinity and in vitro potency of the AB7 antibody for human IL-1β, ascompared to mouse IL-1β. In addition, the data indicate that thisantibody may be used for mouse in vivo disease models with anappropriate higher dose than would be needed for treatment of humansubjects, where the antibody has far superior affinity and potency. Inthe case of other IL-1β antibodies, such as for example other antibodiesdisclosed and/or cited herein, that do not exhibit significantly loweraffinity and in vitro potency for mouse IL-1β, dose adjustments tohigher levels in mouse models may not be necessary.

Example 3 Pharmacokinetics of an Anti-IL-1β Antibody FollowingAdministration of a Single Intravenous or Subcutaneous Dose to Rats

To examine the pharmacokinetic profile, an IL-1β antibody designated AB7was administered to adult male rats as an intravenous (IV) bolus intothe tail vein at doses of 0.1, 1.0, or 10 mg/kg (Groups 1, 2, and 3respectively) or a subcutaneous (SC) dose between the shoulder blades at1.0 mg/kg (Group 4). Blood samples were collected via the jugular veincannula or the retro-orbital sinus at specified times for up to 91 daysafter dosing. Blood samples were centrifuged to obtain serum. Sampleswere analyzed for the concentration of anti-IL-1β antibody using analkaline phosphatase-based ELISA assay as follows.

IL-1β (Preprotech) was diluted to 0.5 μg/mL in PBS and 50 μL of thissolution was added to wells of Nunc-Immuno Maxisorp microtiter plates(VWR) and incubated overnight at 2-8° C. The antigen solution wasremoved and 200 μL of blocking buffer [1% bovine serum albumin (BSA) in1×PBS containing 0.05% Tween 20] was added to all wells and incubatedfor 1 hour at room temperature. After blocking, the wells were washedthree times with wash buffer (1×PBS, containing 0.05% Tween 20).Standards, samples and controls were diluted in sample diluent (25% RatSerum in 1×PBS containing 1% BSA and 0.05% Tween 20). Anti-IL-1βantibody standard solutions were prepared as serial two-fold dilutionsfrom 2000 to 0.24 ng/mL. Each replicate and dilution of the standards,samples and controls (50 μL) were transferred to the blocked microtiterplates and incubated for 1 hour at 37° C. After incubation, the wellswere washed 3 times with wash buffer. Alkaline phosphatase conjugatedgoat anti-human IgG (H+L) antibody (Southern Biotech Associates Inc,Birmingham, Ala.) was diluted 1/1000 in conjugate diluent (1% BSA in1×PBS containing 0.05% Tween 20). Fifty μL of the diluted conjugate wasadded to all wells except for the BLANK wells, which received 50 μL ofconjugate diluent only. The plates were incubated for 1 hour at 37° C.and then all wells were washed 3 times with wash buffer and 3 times withdeionized water. The substrate p-nitrophenylphosphate (1 mg/mL in 10%diethanolamine buffer, pH 9.8) was added to all wells and colordevelopment was allowed to proceed for 1 hour at room temperature, afterwhich 50 μL of 1 N NaOH was added to stop the reaction. The absorbanceat 405 nm was determined using a SPECTRAmax M2 Plate Reader (MolecularDevices, Menlo Park, Calif.) and a standard curve was then plotted asA₄₀₅ versus ng/mL of antibody standard. A regression analysis wasperformed and concentrations were determined for samples and controls byinterpolation from the standard curve. The limit of quantification was40 ng/mL.

As shown in FIG. 3, serum concentrations declined bi-exponentially amongthe IV dose groups. A compartmental analysis was performed on theindividual animal data, and resulting pharmacokinetic parameters wereaveraged for each dose group excluding those animals in which a RAHAresponse was generated. The serum levels of anti-IL-1β antibody declinedwith an average alpha phase half-life of 0.189±0.094 to 0.429±0.043 days(4.54 to 10.3 hours) and a beta phase half-life of 9.68±0.70 to 14.5±1.7days. Among rats receiving a 1 mg/kg subcutaneous dose of AB7 serumlevels increased to a peak of 4.26±0.065 μg/mL by 2-3 days, and declinedwith a half-life of 2.59±0.25 days.

Example 4 Pharmacokinetics of an Anti-IL-1R Antibody FollowingAdministration of a Single Intravenous Dose to Cynomolgus Monkeys

Adult male and female cynomolgus monkeys received the anti-IL-1βantibody designated AB7 as an intravenous (IV) single bolus injection atdoses of 0.3, 3.0, or 30 mg/kg. Blood samples were collected fromanimals prior to dose, 5 minutes, 4 and 8 hours post dose on Day 1, andDays 2, 4, 8, 11, 15, 22, 29, 43 and 56. Samples were analyzed for theconcentration of anti-IL-1β antibody using an alkaline phosphatase-basedELISA assay as follows.

IL-1β solution was diluted to 0.5 μg/mL in PBS and 50 μL of thissolution was added to wells of Nunc-Immuno Maxisorp microtiter plates(VWR) and incubated overnight at 2-8° C. The antigen solution wasremoved and 200 μL of blocking buffer [1% bovine serum albumin (BSA) in1×PBS containing 0.05% Tween 20] was added to all wells and thenincubated for 1-4 hours at room temperature. After blocking, the wellsof each plate were washed three times with wash buffer (1×PBS,containing 0.05% Tween 20). Standards, samples, and controls werediluted in sample diluent (2% Normal Cynomolgus Serum (NCS) in 1×PBScontaining 1% BSA and 0.05% Tween 20). Anti-IL-1β standard solutionswere prepared as serial two-fold dilutions from 8000 ng/mL. Eachreplicate and dilution of the standards, samples, and controls (50 μL)were transferred to the blocked microtiter plates and incubated for 1hour at 37° C. After the primary incubation, the wells were washed 3times with wash buffer and 50 μL of biotinylated rhIL-1 beta was addedto all wells. The plates were then incubated for 1 hour at 37° C. Thewells were washed 3 times with wash buffer and a tertiary incubationwith fifty pL of diluted alkaline phosphatase conjugated streptavidinwas added to all wells except for the BLANK wells, which received 50 μLof diluent only. The plates were incubated for 30 minutes at 37° C., andthen all wells were washed 3 times with wash buffer and 3 times withdeionized water. The substrate p-nitrophenylphosphate (1 mg/mL in 10%diethanolamine buffer, pH 9.8) was added to all wells. Color developmentwas allowed to proceed in the dark for 1 hour at room temperature, afterwhich 50 μL of 1 N NaOH was added to stop the reaction. The absorbanceat 405 nm was determined for all wells using a SPECTRAmax M2 PlateReader (Molecular Devices, Menlo Park, Calif.). A standard curve wasthen plotted as A₄₀₅ versus ng/mL of anti-IL-1β standard. A 4-parameterregression analysis was performed and concentrations were determined forsamples and controls by interpolation from the standard curve. The limitof quantification was 40 ng/mL.

For the single dose 0.3 and 3 mg/kg groups, the serum anti-IL-1βantibody levels declined with an average alpha phase half-life of9.40±2.00 hours, followed by a beta phase half-life of 13.3±1.0 days(FIG. 5). In cynomolgus monkeys receiving a single IV injection of 30mg/kg, serum levels of antibody declined more rapidly, with alpha phasehalf life of 10.9±3.2 hours, followed by a beta phase half-life of7.54±1.79 days. Modeling of plasma concentration-time profiles of 0.1,0.3, 1 and 10 mg/kg doses administered at five monthly intervals alsowas performed and is shown in FIG. 5.

Example 5 Inhibition of Cytokine Production in Human Whole Blood by anIL-1β Antibody

Measuring cytokines in blood during a disease or the treatment of adisease can be useful for determining disease severity or response to atherapy. Usually, cytokine levels are measured in serum, but this methoddoes not necessarily measure total cytokines. Many cytokines can beinside cells (intracellular). In addition, the ability for a cell toproduce a cytokine may be more useful information than the level ofcirculating cytokine.

A method of stimulating whole blood was used to determine cytokineproduction and the effect of treating with an anti-IL-1β antibody. Bloodwas drawn from patients into sterile heparinized tubes and then 250 ulof the whole blood was added to each 4 mL orange top Corning sterilecryotube set up as follows:

Control Series

All tubes were pre-filled with 550 ul of RPMI. To tube 1 (control), 200ul RPMI was added and to tubes 2-10, 100 ul additional RPMI was added.To each of tubes 2-10, 100 ul of dilutions of an anti-IL-1β antibody(AB7) was added.

Test Series

A similar series of antibody dilutions was set up as detailed above.

All tubes were mixed well using a 10 second vortex. Control series tubesA1-10 then received an additional 100 ul of RPMI, were vortexed 10seconds, the screw cap tightly fixed and the tubes placed in incubator.To Test series tubes B1-10, 100 ul of heat-killed Staphylococcusepidermidis (final concentration of 1:1000 of stock resulting in abacterium:white blood cell ration of 10:1) was added, the tubes werethen vortexed for 10 seconds, capped and placed in 37° C. incubator.After 24 hours incubation, the cultures were all lysed with Triton X(0.5% final) to release the cell contents and the lysates were frozen.After lysis of the whole blood cultures, the tubes subjected to freezethaw cycles and cytokine levels are measured by standard cytokine ELISAassays for human TNFα, IL-6, IFNγ, IL-8, IL-1α, IL-1Ra and IL-1β (R&DSystems, Minneapolis, Minn.).

Cytokines measured in the control series tubes, which contain onlysterile culture medium and antibody (where indicated), reflect thespontaneous level of stimulation. In healthy subjects, very low levelsof the various cytokines are found when measured after 24 hours ofincubation. In patients with untreated diseases, the levels may behigher. The Test series of tubes additionally contained a defined amountof heat-killed Staphylococcus epidermidis, which stimulates productionof a number of cytokines. If the anti-IL-1β antibody treatment isefficacious, this will be reflected by reduces cytokine production.

As shown in FIG. 6, the high affinity anti-IL-1β antibody AB7 was veryeffective at inhibiting the production of IL-1β in human blood. In anaverage of three human samples, the antibody inhibited the production ofIL-1β induced by Staphylococcus epidermidis by 50% at 0.1 pM and by 75%at 3 pM. At 100 pM, inhibition was 100%. Interferon gamma (IFNγ) wasinduced by Staphylococcus epidermidis and AB7 reduced IFNγ induced byStaphylococcus epidermidis by 75% at 100 pM.

Example 6 Pharmacokinetics of an Anti-IL-1β Antibody FollowingAdministration of a Single Intravenous Dose to Humans

Pharmacokinetics of an IL-1β antibody having the aforementionedproperties was demonstrated in a phase I human clinical study.Specifically, a double-blind, placebo controlled human clinical studywas performed in Type 2 diabetes patients and data initially obtainedfrom five patients receiving the IL-1β antibody designated AB7(described above) at a dose of 0.01 mg/kg via constant rate intravenousinfusion were used to examine pharmacokinetics.

On study Day 1, antibody was administered either via a 30 minuteconstant rate intravenous infusion. Safety assessments, including therecording of adverse events, physical examinations, vital signs,clinical laboratory tests (e.g., blood chemistry, hematology,urinalysis), plasma cytokine levels, and electrocardiograms (ECGs) wereconducted using standard medical practices known in the art. Bloodsamples were collected pre-dose administration and at days 0, 1, 2, 3,4, 7, 9±1, 11±1, 14±1, 21±2, 28±2, 42±3, and 56±3 post-administration toassess IL-1β antibody levels (pharmacokinetics). Preliminary analysis ofthe pharmacokinetics of the IL-1β antibody in subjects receiving asingle IV dose of 0.01 mg/kg showed serum concentration-time profileswith a terminal half-life of 22 days, clearance of 2.9 mL/day/kg andvolume of distribution of the central compartment of 50 mL/kg, verysimilar to serum volume (FIG. 7).

Interim analysis of pharmacokinetic data following IV administration ofa single dose of AB7 (XOMA 052) in subjects through the 0.01, 0.03, 0.1,0.3, or 1.0 mg/kg dose groups further confirmed that the serumconcentration-time profiles with a terminal half-life of 22 days,clearance of 2.54 mL/day/kg and volume of distribution of the centralcompartment of 41.3 mL/kg, very similar to serum volume (FIG. 8).

Example 7 Effects of an IL-1β Antibody on CRP in Human Subjects withType 2 Diabetes

C-reactive protein (CRP), which is released by the liver in response tovarious stress triggers, including IL-6, produced in response to IL-1,also was measured in serum at the same time points as the PK samples todetermine the activity of the antibody in human subjects. A single doseof XOMA 052 reduced ultra-sensitive C-reactive protein (usCRP) levels, astandard measure of systemic inflammation associated with multiplediseases and an indicator of cardiac risk, in all of the dose groupstreated compared to placebo. As shown in FIG. 9, at 28 days after asingle dose of XOMA 052, the median percent reductions in usCRP were 33,46, 47, 36, and 26 for the 0.01, 0.03, 0.1, 0.3, and 1.0 mg/kg dosegroups, respectively, compared to 4 percent for placebo. The activityresulting from a single administration of antibody at a dose of 0.01mg/kg indicates that even lower doses may be used.

Example 8 Use of an IL-1β Antibody in the Treatment of Gout in an AnimalModel

Efficacy of an IL-1β antibody, such as an antibody having theaforementioned properties or as described herein, was evaluated in anacute mouse model of gout. The acute mouse model of gout evaluates theability of a therapeutic agent to block monosodium urate (MSU)crystal-induced acute peritonitis (Martinon et al., 2006, Nature440:237-241). Specifically, peritonitis was induced by injecting 0.5 mgof MSU crystals into the peritoneal space of Balb/c mice. Mice weretreated 2 hours earlier by intraperitoneal injection of isotype controlantibody or anti-IL-1β antibody XOMA 052 (also referred to as AB7herein, described above) at 10 mg/kg. For comparison, one group of micereceived Anakinra at 30 mg/kg at the same time as MSU injection. After 6hours, peritoneal lavage was performed and the lavage fluid wascentrifuged to collect cells. Cells were counted and a fraction was usedfor cytospin and leukocyte differential counts. Peritonitis was measuredby calculating the number of neutrophils in the lavage. The number ofneutrophils is determined by multiplying the total cell count in thelavage by the percentage of neutrophils in the differential count. Asshown in FIGS. 8A and 8B, the XOMA 052 antibody was able to block theneutrophil and macrophage infiltration, and reduce peritonitis inducedby the MSU crystals relative to the PBS and isotype controls (p<0.05,unpaired t-test). There was no significant difference between treatmentwith 10 mg/kg XOMA 052 and 30 mg/kg Anakinra in the mouse model.

Example 9 Use of an IL-1β Antibody in the Treatment of Gout

IL-1β antibodies or fragments, such as those having the aforementionedproperties or described herein, may be administered to a subject (e.g.,human patient) for therapeutic treatment and/or prevention of gout.Specifically, in one example, an IL-1β antibody XOMA 052 (also known asAB7, described above) is used for the therapeutic treatment of patientsdisplaying signs and symptoms of gout. Safety and effectiveness of theIL-1β antibody for gout are demonstrated in one or more human clinicalstudies, including for example a trial of the following design insubjects with recurrent acute gout.

Subjects may be included in the study if they meet all of the followingcriteria:

-   -   Acute gout diagnosed by meeting criteria from the 1977 Criteria        for the Classification of Acute Arthritis of Primary Gout        (American Rheumatism Association, ACR). A diagnosis of acute        gout is confirmed by a) the presence of characteristic urate        crystals in the joint fluid; b) a tophus proven or suspected to        contain urate crystals by polarized light microscopy; or c) the        presence of at least 7 of the following 12 clinical, laboratory,        or radiographic phenomena:    -   More than one lifetime attack of acute arthritis    -   Maximum inflammation developed within 1 day    -   Attack of monoarticular arthritis    -   Observed joint redness    -   First metatarsophalangeal (MTP) joint painful or swollen    -   Unilateral first MTP joint attack    -   Unilateral tarsal joint attack    -   Tophus (proven or suspected)    -   Hyperuricemia    -   Asymmetric swelling within a joint on X-ray/exam    -   Subcorticol cysts without erosions on X-ray    -   Joint fluid culture negative for organisms during attack    -   At least two acute gout attacks within the previous year    -   Onset of the current acute episode must have occurred no more        than 48 hours prior to study drug administration on Day 0 and        the symptoms of the acute attack must not have significantly        subsided prior to study drug dosing

A phase 1/2, double-blind, placebo-controlled, parallel-group,single-dose study of the safety and pharmacokinetics of XOMA 052antibody performed in subjects experiencing acute gout attacks. Subjectsin parallel dose groups of six subjects each (multiple active druggroups and one placebo group) are enrolled to receive a single IVinfusion of study drug (IL-1β antibody or placebo) at dose levels shownin the table below.

Dose Number Group of Subjects Dose Regimen A 6 Single IV infusion ofantibody at 0.03 mg/kg B 6 Single IV infusion of antibody at 0.1 mg/kg C6 Single IV infusion of antibody at 0.3 mg/kg D 6 Single IV infusion ofantibody at 1.0 mg/kg E 6 Single IV infusion of antibody at 3.0 mg/kg F6 Single IV infusion of placebo

Subjects that meet all eligibility criteria are enrolled, randomizedinto one of the dose groups, and dosed on Day 0 with study drug(antibody or placebo). Subjects must be dosed within 48 hours of theonset of the new acute gout attack. A new attack is defined as one thatfollows at least 28 days in which the subject is free of acute goutsymptoms.

Weekly assessments are performed through Day 28, followed by biweeklyassessments through Day 56. Safety is assessed by pre- andpost-treatment serial measurements of vital signs, clinical laboratoryassessments, and the recording of adverse clinical events. PK data iscollected and analyzed at the time points shown below.

Pharmacokinetic Assessment

Serum samples are collected on Day 0 (baseline) prior to study drugadministration, and at selected time points after the administration ofthe study drug for the measurement of serum concentrations of IL-1βantibody.

From these serum concentrations, the appropriate pharmacokineticparameters are calculated. These calculations are expected to employcompartmental and noncompartmental pharmacokinetic methods to estimatethe following parameters: peak concentration, serum clearance,half-lives, volumes of distribution, and mean residence time. PopulationPK and PD analysis methods may be employed to better understand thePK/PD characteristics of the IL-1β antibody in this population.

The IL-1β antibody pharmacokinetics are evaluated for their correlationwith biological markers and clinical outcome. In addition, an assessmentis made of the correlation between drug exposure and any evidence ofdrug toxicity.

Biological and Clinical Activity

As measures of the biological activity of IL-1β antibody in subjectswith acute recurrent gout, CRP and ESR are collected as inflammatorymarkers. The clinical status of the gout, including pain level and timecourse, is measured through periodic physician assessments and subjectself-assessments. Clinical assessment of the acute gout episode includesa physician's assessment of redness, tenderness, and swelling (none ofwhich are attributable to other causes), a physician's globalassessment, and a subject pain self-assessment, a patient's globalassessment, and a HAQ. Subjects are given a diary and asked to report ontheir symptoms every 2 hours during waking time in the first 24 hourspost-dose, followed by two times per day through Day 7, once per daythrough Day 14, and only as symptoms recur thereafter.

The clinical assessment of acute gout includes the following components:

Physician's Assessments:

-   -   Physician's Global Assessment (10-point analog scale)    -   Physician's assessment of erythema (10-point analog scale)    -   Physician's assessment of heat (10-point analog scale)    -   Physician's assessment of swelling (10-point analog scale)

Subject's Assessments:

-   -   Patient's Global Assessment (10-point analog scale)    -   Pain at rest (10-point analog scale)    -   Pain on weight-bearing/movement (10-point analog scale)    -   Health Assessment Questionnaire (HAQ)

In addition, the following assessments are performed at various samplecollection time points:

-   -   Serum collected at various time points is used to assess levels        of adipokines and cytokines that may be produced by inflamed        adipose tissue in gout patients. Such adipokines/cytokines        include, for example, adiponectin, resistin, leptin, visfatin,        PAI 1, TNFα, IL-1, IL-1Ra, IL-6, IL-8, RANTES, and MCP-1.    -   A whole blood sample is collected and assayed for cytokines that        may include, for example, TNFα, IL-6, IFNγ, IL-8, and IL-1α.

PK Sampling Schedule Time Point All Groups Day 0 Prior to infusion X EOIX EOI + 30 ± 5 min X EOI + 4 hr ± 15 min X Day 1 (24 ± 2 hr post EOI) XDay 2 (48 ± 2 hr post EOI) X Day 3 (72 ± 3 hr post EOI) X Day 4 (96 ± 3hr post EOI) X Day 7 X Day 9 X Day 11 X Day 14 X Day 21 X Day 28 X Day42 X Day 56 X ¹ Adipokines/cytokines are analyzed using samplescollected at some or all of the PK time points. Measuredadipokines/cytokines may include, for example, adiponectin, resistin,leptin, visfatin, PAI-1, TNFα, IL-1, IL-1Ra, IL-6, IL-8, RANTES, andMCP-1. EOI = end of infusion

Based on results obtained from the first clinical study, additionalclinical trials are performed. Such trials may include one or more ofthe above dosages and dosing regimens, as well as or alternatively oneor more other dosages of IL-1β antibody, longer treatment and/orobservation periods and greater numbers of patients per group (e.g., atleast about 10, 50, 100, 200, 300, 400, 500, 750, 1000)

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Wherever an open-ended term isused to describe a feature or element of the invention, it isspecifically contemplated that a closed-ended term can be used in placeof the open-ended term without departing from the spirit and scope ofthe invention. Recitation of ranges of values herein are merely intendedto serve as a shorthand method of referring individually to eachseparate value falling within the range, unless otherwise indicatedherein, and each separate value is incorporated into the specificationas if it were individually recited herein. All methods described hereincan be performed in any suitable order unless otherwise indicated hereinor otherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseworking in the art upon reading the foregoing description. The inventorsexpect skilled artisans to employ such variations as appropriate, andthe inventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

1. A method of treating gout in a subject, the method comprisingadministering an anti-IL-1β antibody or fragment thereof to the subject.2. The method of claim 1, wherein the gout is chronic gout.
 3. Themethod of claim 1, wherein the gout is acute gout. 4-59. (canceled)